Identification of lncRNA-Binding Proteins

The biotin-labeled lncRNA probe (PSMA3-AS1, 5’- TACAAAATCCATCTGCT GACCCCTG-biotin-TEG-3′; MIR22HG, 5′-TCTCTACCACTGTCCCACACGCATC-biotin-TEG-3′; biotin-TEG, biotin with 15 atom triethylene glycol spacer) was synthesized by Hangzhou Youkang Biotechnology Inc. The lncRNA-biotin probe was incubated with streptavidin magnetic beads (Beyotime) in 1 × Binding & washing buffer (10 mM Tris–HCl, 1 mM EDTA, 2 M NaCl, 0.01–0.1% Tween-20, pH 7.5) for 30 min at room temperature. Then the lysate of cells were incubated with washed beads in for 2 h at 4 °C. Subsequently, miRNAs or proteins were extracted from the beads. The miRNA extraction was performed using mirVana miRNA Isolation Kit (Thermo Fisher Scientific) and analyzed by qPCR. The proteins were eluted using 0.1% SDS, followed by SDS-PAGE. Mass spectrometry was used for identification of binding proteins.