The biotinylated probes for hsa_circ_0050386 and the negative control (NC) probes were designed and synthesized by RiboBio (Guangzhou, China). hsa_circ_0050386 probe sequences are listed in Additional file 3 : Table S2. The protocol for the RNA pull-down assay was performed based on those previously reported [18 (link)].
1 × 107 cells were washed in ice-cold PBS and lysed in 500 μl of co-IP buffer. After that, 3 μg of biotinylated probes targeting the hsa_circ_0050386 back splicing junction region or control probe were added to the cell lysates. The probes in the cell lysates were incubated for 2 h at room temperature. Each binding reaction was then treated with 50 μl of washed Streptavidin C1 magnetic beads (Invitrogen) and left to incubate for one hour at room temperature. The magnetic beads underwent a brief wash cycle of five times using Co-IP buffer. The proteins that were retrieved will be used for mass spectrometry or Western blot analyses.
1 × 107 cells were washed in ice-cold PBS and lysed in 500 μl of co-IP buffer. After that, 3 μg of biotinylated probes targeting the hsa_circ_0050386 back splicing junction region or control probe were added to the cell lysates. The probes in the cell lysates were incubated for 2 h at room temperature. Each binding reaction was then treated with 50 μl of washed Streptavidin C1 magnetic beads (Invitrogen) and left to incubate for one hour at room temperature. The magnetic beads underwent a brief wash cycle of five times using Co-IP buffer. The proteins that were retrieved will be used for mass spectrometry or Western blot analyses.