Ez magna riptm kit

RIP assay was designed according to MS2bp-MS2bs system according to a previous study 48 (link). Briefly, 293T cells were seeded in cultural plates for 24 h, and then co-transfected with 30 μg pcDNA3.1-MS2bs plasmid including Nanog, SOX2, OCT4, SATB2, Renilla luciferase (as blank control vectors), 10 μg MS2bp-GFP plasmid and miR143/145 mimics by Lipofectamine 2000 (Invitrogen). After 48 h, cells were detected for RNA immunoprecipitation using GFP antibody based on EZ-Magna RIP^TM Kit (Millipore). The complexes of purified RNA were performed for RT-PCR and co-isolated RNA-binding proteins were detected by Western blot.

RNA-binding protein immunoprecipitation (RIP) assays were performed using the EZ-Magna RIP^TM Kit (No.17-701; Millipore) in accordance with the manufacturer’s instructions. In brief, around 2 × 10⁷ cells were lysed in RIP lysis buffer provided in the Kit, subsequently, the lysates were incubated with Anti-Flag M2 magnetic beads (No.8223; Sigma) at 4 °C for overnight, and then the precipitate complexes were washed with washing buffer for six times and treated with proteinase K. Immunoprecipitated RNA was extracted using the phenol/chloroform method, and the RNA was subjected to reverse transcription and qPCR analysis. qPCR primers were listed in Supplementary Table 1.

The RIP assay was conducted to verify the interaction of AGO2 proteins with circPOSTN or STC1 RNAs using the EZ-Magna RIP^TM Kit (Millipore, MA, USA) under the manufacture’s instruction. 2 × 10⁷ GBM cells were lysed in RIP lysis buffer and incubated magnetic beads conjugated to 5 μg anti-AGO2 antibodies (Abcam) or 5 μg normal anti-IgG antibodies at 4 °C overnight to capture the RNAs bound to AGO2 proteins. Then, the captured RNAs were extracted from the beads and submitted to qPCR analysis.