Argonaute-2 Immunoprecipitation Protocol

The RIP assay was performed in this study using the Magna RIP RNA-Binding Protein IP Kit (Millipore, Burlington, MA, USA) according to the product instruction. Cells were collected and washed twice with pre-cooled PBS and centrifuged at 1500 rpm for 5 min at 4 °C to discard supernatant, followed by adding RIP Lysis Buffer to lyse the cells on ice for 5 min. Diluted 50 µL of magnetic beads in 100 µL of RIP Wash Buffer was used. The sample was incubated with 5 µg of Argonaute-2 antibody or 1 µg IgG antibody for 30 min at room temperature with rotation. After light centrifugation, the supernatant was removed, and the precipitate was resuspended in 0.5 mL RIP Wash Buffer. After that, 900 µL of RIP Immunoprecipitation Buffer was added to the bead-antibody mixture and then incubated with 100 cell lysis products overnight at 4 °C with rotation. Finally, 150 µL proteinase K buffer was added to the complex product and incubated at 55 °C for 30 min, followed by RNA extraction for RT-qPCR.