Biotin-RNA Interactome Profiling

The assays were performed by the method as previously described^{15 (link)}. Briefly, for each target mRNA fragment, template DNA was synthesized by PCR with gene specific primer sets containing T7 RNA polymerase promoter sequence. Biotinylated RNA fragments were generated by in vitro transcription using MAXIscript T7 Transcription Kit (Thermo Fisher Scientific) and Biotin-11-CTP (PerkinElmer, Waltham, CT, USA). After incubation of 2.5 μg biotinylated RNA with 80 μg of whole cell lysates in TENT buffer (10 mM Tris-HCl buffer, pH 8.0, 250 mM NaCl, 0.5% Triton X-100, and 1 mM EDTA) for 30 min, Dynabeads M-280 Streptavidin (Thermo Fisher Scientific) were added and incubated at room temperature for 1 h. The proteins bound to the beads were isolated with magnetic beads separator and followed by western blot assay or liquid chromatography-mass spectrometry assay (nanoLC-MS/MS, CapLC, Q-Tof).