Rip lysis buffer

RIP assay was performed using the Magna RIPTM RNA Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA, USA) according to the manufacturer’s protocol. Briefly, cultured chondrocytes were collected and resuspended in RIP lysis buffer (Solarbio) then the cell extracts were incubated with RIP buffer containing magnetic beads conjugated with human anti-Ago2 antibody (Millipore) or mouse immunoglobulin G (IgG) control overnight at 4 °C. The next day, the magnetic beads were incubated with proteinase K after washing three times. Total RNAs was isolated from the extracts using the TRIzol reagent subsequently. Lastly, the relative enrichment of HOTAIR and miR-17-5p were determined by RT-qPCR analysis.

RIP was performed using a Magna RIPTM RNA kit (Millipore, USA). Briefly, cultured chondrocytes were suspended in RIP lysis buffer (Solarbio) and incubated in RIP buffer containing human anti-Ago2 antibody beads (Millipore) overnight (Input and normal IgG served as controls). Next, RNAs were extracted using TRIzol reagent to follow the relative enrichment of MEG3/FOXO1 and miR-361-5p.

RNA immunoprecipitation (RIP) assay was performed by Imprint RNA immunoprecipitation kit (Sigma–Aldrich) according to manufacturer’s instructions. Briefly, IgG-induced Hela cells were collected and resuspended in RIP lysis buffer (Solarbio), subsequently centrifuged at 12000×g for 5 min. Then, cell lysate was incubated with anti-Argonaute2 (anti-Ago2) or anti-IgG (NC) overnight at 4°C, followed by the addition of Protein A magnetic beads to get the immunoprecipitation complex. At last, the relative enrichment of RP11-284F21.9 and miR-769-3p were detected by RT-qPCR.

RNA immunoprecipitation assay was performed by Imprint RNA immunoprecipitation kit (Sigma-Aldrich) referring to the recommended protocols of manufacturer. Firstly, IL-1β-induced chondrocytes were collected and resuspended in RIP lysis buffer (Solarbio), subsequently centrifuged at 12,000g for 5 min. Then, cell lysate was incubated with anti-Argonaute2 (anti-Ago2) or anti-IgG (negative control) overnight at 4 °C, followed by the addition of Protein A magnetic beads to get the immunoprecipitation complex. Total RNA was isolated using GenElute™ Total RNA Purification Kit (Sigma-Aldrich). Lastly, the relative enrichment of MEG3 and miR-16 were determined by RT-qPCR analysis.