Riptm rna binding protein immunoprecipitation kit

RIP was conducted in SiHa cells with magna RIPTM RNA-binding Protein Immunoprecipitation kit (Millipore, Billerica, MA). SiHa cells were transfected with miR-320a mimics, and then lysed in complete RNA lysis buffer after 48 h. Cell lysates were rotated in RIP immunoprecipitation buffer including magnetic beads conjugated with negative control mouse IgG or human anti-AGO2 antibody (Mouse, Millipore, Billerica, USA) overnight. Next day, immunoprecipitated RNA was extracted after incubating with Proteinase K for 30 min. Last, qRT-PCR and agarose gel electrophoresis were performed to identify the expression of circCLK3 and miR-320a.

The RIP experiment was performed using a RIPTM RNA-binding protein immunoprecipitation kit (Millipore, USA) according to the manufacturer’s instructions. Approximately 5 μg of antibody (target protein, FOXF2; negative control protein, rabbit IgG; positive control protein, SNRNP70) was added, and the sample was incubated with protein G magnetic beads. After the addition of cell lysis buffer, the coprecipitated RNA was pulled down with protein G beads, followed by high-throughput sequencing.

RIP assay was conducted with magna RIPTM RNA-binding Protein Immunoprecipitation kit (Millipore, Billerica, MA). MKN-28 cells were transfected with miR-1306-3p mimics or negative control. The cells were lysed in complete RNA lysis buffer after 48 h. Then, the RIP immunoprecipitation buffer including magnetic beads conjugated with negative control mouse IgG or human anti-AGO2 antibody (Mouse, Millipore, Billerica, USA) was added into cell lysates. Subsequently, the lysates were rotated overnight. Next day, after incubating with Proteinase K for 30 min, the immunoprecipitated RNA was extracted. Last, qRT-PCR and agarose gel electrophoresis were performed to identify the expression of circNHSL1 and miR-1306-3p.