Myone dynabead streptavidin c1 beads

For biotinylated RNA pull-down assays, transfected HepG2-SR cell lysates were incubated with MyOne™ Dynabead® Streptavidin C1 beads (Invitrogen, USA) at 4 °C for 1 h. For biotinylated-probe pull-down assays, MyOne™ Dynabead® Streptavidin C1 beads were incubated with HepG2-SR cell lysate at 4 °C for 1 h for preclearance. The 3′ biotin-labeled circRNA-SORE probe (5′-GAGTTGTTGCTGCTTGATGGAGTC-3′-biotin) was synthesized by Tsingke Biotech (Beijing, China) and incubated with the beads at room temperature for 10 min for immobilization. The biotinylated beads were incubated with HepG2-SR cell lysates at 4 °C overnight. The beads were then magnetically separated and washed five times. For western blot detection, the beads were boiled in SDS buffer for protein/peptide isolation. For mass spectrum assays, the beads were incubated with non-ionic water in 70 °C for 5 min.

Biotinylated-probe pull-down assays were performed in HCCLM3. The cells were lysed with a mixture of protein and IP buffer (Beyotime, China), along with protease inhibitor (Medchemexpress, USA), and RNase inhibitor (Sigma, USA). After prewash, the HCCLM3 cell lysates were incubated with MyOne™ Dynabead® Streptavidin M270 beads (Invitrogen, USA) at 4°C overnight. The 5' biotin-labeled cDCBLD2 probe (5'-biotin-gctaTCCACATCCATCACCTTGTTTATCTATA-3') or its control (5'-biotin-gctaTATAGATAAACAAGGTGATGGATGTGGA-3') was synthesized by Tsingke Biotech (Beijing, China) and incubated together. For miRNA biotinylated-probe pull-down assays, the 3' biotin-labeled miRNA345-5p probe (5'-GCUGACUCCUAGUCCAGGGCUC-3'-biotin) and its negative control were transfected into the cell in advance. Then the cells were lysed by the above method and lysates were incubated with MyOne™ Dynabead® Streptavidin C1 beads (Invitrogen, USA) at 4°C overnight. Collect the beads and wash several times, then use Trizol for RNA extraction.