Probes streptavidin dynabeads m 280

CircRIP assay was performed using a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) as described in our previous study [8 (link)]. Biotin-labeled circ_0020710 (Tables S4) was synthesized by Sangon Biotech (Shanghai, China). Briefly, circ_0020710-overexpressing cells were washed with PBS, fixed in 1% formaldehyde, lysed in Co-IP buffer, sonicated and finally centrifuged. The supernatant (50 μL) was used as input and the rest was incubated with a probes-streptavidin-dynabeads (M-280; Invitrogen, Carlsbad, CA, USA) mixture at 30 °C for 12 h. The mixture was incubated with lysis buffer and proteinase K. Then the RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and used for further studies.

RNA immunoprecipitation (RIP) assay was conducted using a Magna RIP RNABinding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA), following the procedures outlined in our previous study (Zhang et al. 2019 (link)). Biotin-labeled circFCHO2 was synthesized by Geneseed Biotech (Guangzhou, China). In brief, circFCHO2-overexpressing cells were washed with PBS, fixed in 1% formaldehyde, lysed in Co-IP buffer, subjected to sonication, and finally centrifuged. A portion of the supernatant (50μL) was reserved as input, while the remaining supernatant was incubated with a mixture of probes-streptavidin-dynabeads (M-280; Invitrogen, Carlsbad, CA, USA) at 30℃ for 12 h. The mixture underwent further incubation with lysis buffer and proteinase K. Subsequently, RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA), eluted, reverse transcribed to cDNA, and subsequently detected by qRT-PCR.