The RNA pulldown assay was performed as previously described with minor modification [27 (link)]. Biotin-labelled circ-AKT3 probe was synthesized by Tsingke (Beijing, China), the sequence of the probe was just complemented to the back-spliced junction of circ-AKT3(listed in Additional file 1 : Table S1). Circ-AKT3 overexpressing OSRC-2 cells were scratched, lysed, and sonicated. After centrifugation, 50 μL of the supernatant was retained as input, and the remaining part was incubated with streptavidin dynabeads (M-280, Invitrogen) conjugated with biotin-labelled circ-AKT3 probe overnight at 4 °C. The mixture was washed, and total RNA was then isolated to detect the expression of circ-AKT3 and miRNAs by RT-PCR and qRT-PCR.
The AGO2-RIP assay was performed using the MagnaRIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, MA, USA) as previously described [28 (link)]. After incubating circ-AKT3 overexpressing OSRC-2 cells lysate with dynabeads coated with 5 μg of control rabbit IgG or antibody against Argonaute-2 (AGO2) (Abcam, MA, USA) with rotation at 4 °C overnight, total RNA was retrieved for the detection of circ-AKT3 and miRNA expression by qRT-PCR.
The AGO2-RIP assay was performed using the MagnaRIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, MA, USA) as previously described [28 (link)]. After incubating circ-AKT3 overexpressing OSRC-2 cells lysate with dynabeads coated with 5 μg of control rabbit IgG or antibody against Argonaute-2 (AGO2) (Abcam, MA, USA) with rotation at 4 °C overnight, total RNA was retrieved for the detection of circ-AKT3 and miRNA expression by qRT-PCR.