RNA-Protein Interactome Profiling in Embryos

In all, 8-cell or 1k-cell stage embryos were dechorionated with 1 mg/mL of pronase. RIP was performed as described in Ren et al, 2020 (link) with some modifications. Briefly, embryos were lysed in cold RIP buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 0.5% IGEPAL^® CA-630 (Sigma-Aldrich), 1 mM DTT (Merck), cOmplete, EDTA-free protease inhibitor cocktail (Merck) and 40 U/μL RNAse Out (Invitrogen) and homogenized with a Dounce homogenizer (Sigma-Aldrich). Lysates were cleared at 13,000×g for 10 min at 4 °C and incubated with anti-GFP agarose beads (Molecular Biology Service, IMP), previously equilibrated with RIP buffer for 2 h at 4 °C. Overall, 200 μL of each lysate, corresponding to the input sample, was kept on ice. Beads were washed two times with RIP buffer and two times with washing buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl₂, and 0.5% IGEPAL^® CA-630 (Sigma-Aldrich). Beads were incubated with 0.3 μg/μl proteinase K, 50 mM Tris-HCL pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 0.5% IGEPAL^® CA-630 (Sigma-Aldrich) and 1% SDS for 30 min at 55 °C. RNA from elution and input samples was purified with the RNA Clean & Concentrator kit (Zymo Research) according to the manufacturer’s protocol.