Profiling lnc-AROD Interactions by AGO2 IP and MS2-RIP

For AGO2 immunoprecipitation, lnc-AROD-overexpressing 293T cells were plated into six-well plates in three replicates and transfected with 3×Flag-AGO2 or 3×Flag-GFP for 48 h. The cells were UV cross-linked and lysed with RIPA buffer (10 mM HEPES [pH 7.4], 200 mM NaCl, 30 mM EDTA, and 0.5% Triton X-100) supplemented with RNase (Beyotime, China) and protease inhibitors for 15 min. The lysates were incubated with magnetic beads conjugated with IgG (as a control) and an anti-Flag antibody (F1804; Sigma-Aldrich, St. Louis, MO, USA) at 4°C for 4 h. The beads were washed five times and lysed in TRIzol reagent for RNA extraction and subjected to RT-qPCR to determine the amount of lnc-AROD.
An MS2-RIP assay was performed as previously reported (47 (link)). Briefly, 293T cells were plated into six-well plates in three duplicates and transfected with MS2 or MS2–lnc-AROD together with MS2-GST and miR-324-5p for 48 h. The cells were subsequently lysed with NT2 buffer (50 mM Tris-HCl [pH 7.0], 150 mM NaCl, 1 mM MgCl₂, 0.05% NP-40, 1 mM phenylmethylsulfonyl fluoride, 10 mM ribonucleoside vanadyl complex) supplemented with RNase (Beyotime, China) and protease inhibitors for 15 min. The lysates were incubated with GST-magnetic beads at 4°C for 4 h and washed five times, and RNA was extracted from the beads followed by RT-qPCR to quantify the amount of miR-324-5p.