Magna rip rbp immunoprecipitation kit

RIP experiments were performed using a Magna RIP RBP Immunoprecipitation kit (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. Briefly, the HEK293 cells were washed twice with precooled phosphate-buffered saline (PBS) and lysed with an equal volume of RIP lysis buffer. RIP wash buffer was used to prepare the magnetic beads. Then, 100 µL of cell lysate was added to the magnetic beads and resuspended in 900 µL of RIP buffer, and incubated overnight at 4 °C. The magnetic bead and antibody (or specific probes) complex were resuspended with proteinase K buffer incubated for 30 min at 55 °C and washed in RIP buffer, phenol, and chloroform. Finally, salt solution and precipitate enhancer were added, and anhydrous ethanol was also added and incubated at 80 °C for 1 h. After centrifuging, the precipitate was dissolved in diethylpyrocarbonate for qPCR analysis

Total RNA was extracted from cells using TRIzol reagent (Life Technologies) according to the manufacturer’s instructions. Plasma RNA was extracted using a TIANamp virus RNA kit (Tiangen, Beijing, China). For the detection of RNA stability, total RNA (2 μg) was incubated with 1 μL of RNase R (Epicenter Technologies, Madison, WI, USA) at 37°C for 20 min. Actinomycin D (2 μg/mL) was added to the culture medium, and cells were collected at the appointed time. RIP experiments were performed with a Magna RIP RBP immunoprecipitation kit (Millipore, Bedford, MA, USA) to obtain RNAs, which were used to detect the abundance of circRNAs. RNAs were reverse transcribed using Goldenstar RT6 cDNA synthesis kits (Tsingke, Beijing, China) with random primers or oligo(dT)₁₈ primers. Also, qPCR was performed with Hieff qPCR SYBR Green master mix (Yeasen, Shanghai, China). GAPDH mRNA was used as a control. Primers are listed in Table S1.