Rna pull down kit

The RNA pull-down assay was performed using an RNA pull-down kit (BersinBio, Guangzhou, China) according to the manufacturer's instructions. Biotinylated circRSU1 probe was designed and synthesized by RiboBio (Guangzhou, China). In brief, 1 × 10⁷ human chondrocytes were harvested, lysed, and sonicated. C-1 magnetic beads were incubated with a circRSU1 probe or oligo probe at 25 °C for 2 h to generate probe-coated beads. These probe-coated beads were incubated with the cell lysates at 4 °C overnight to pull down circRSU1. After three sequential washes with the wash buffer, RNA complexes bound to the beads, were extracted using a RNeasy Mini kit (QIAGEN), and analyzed by qRT-PCR.

The binding proteins of LINC00839 were collected using RNA pull down Kit (BersinBio, China). Biotinylated RNA probes were incubated with cell protein extract to form RNA–protein complexes. The complexes were separated from other components in the incubated solution through binding to streptavidin-labeled magnetic beads and the proteins were eluted to further analyzed.

Biotinylated AC006064.4–201 and Gm49317-201 probes were designed and synthesized by RiboBio (Guangzhou, China). An RNA pull-down kit (BersinBio, Guangzhou, China) were used for RNA pull-down assay. Approximately 1 × 10⁷ human or mouse chondrocytes were harvested and lysed. The AC006064.4–201, Gm49317-201 and Oligo probes were added to the magnetic beads. The cell lysates were incubated with these probe-coated beads at 4 °C overnight. The RNA–protein complexes were then eluted for western blotting analysis. The biotinylated probes used in this study are listed in Supplementary Table S4.

Biotin-labeled circTTN probe and NC probe were provided by Ribo Biotechnology (Guangzhou, China). Pull-down protein experiments were performed using an RNA pulldown kit (BersinBio, Guangzhou, China). First, the circTTN probe and NC probe formed a secondary structure and were incubated with streptavidin magnetic beads, respectively. Then, 2 × 10⁷ cell samples were collected for protein extraction and the nucleic acid of protein samples were removed, and then, the probe–magnetic bead complex was rotatively incubated with cell extracts. Finally, the histone samples of the circTTN group and NC group were collected, and silver dyeing was performed with a silver dyeing kit (Beyotime, Shanghai, China). First, 30% ethanol was used for fixing, and then, silver sensitizing solution, silver solution, silver dyeing solution, and silver dyeing stop solution were added successively. The silver dyed adhesive strips were photographed and the results were analyzed. RNA pulldown protein samples were determined and analyzed using mass spectrometry in Genecreate Biotechnology (Wuhan, China).

The RNA pulldown assay was performed in NB4 cells using the RNA pulldown Kit (BersinBio, Guangzhou, China) according to the manufacturer’s instruction. Briefly, the biotin-labeled circSPI1 probe or negative control probe was incubated with streptavidin magnetic beads. A total of 2 × 10⁷ NB4 cells were collected and lysed in RIP buffer to obtain whole protein extract. The probe magnetic beads and cell extract were mixed and washed with NT2 buffer. Protein bound with the probe was eluted with the protein elution buffer and subjected to western blotting.

The biotin-labeled miRNA pull-down assay was performed using the RNA Pulldown Kit (BersinBio, Guangzhou, China). Briefly, cells were transfected with biotin-labeled miR-195-3p and miR-ctrl (RiboBio, 50 nM), and lysed after 48 h incubation. Simultaneously, 25 μg streptavidin magnetic beads were mixed with sample lysates and incubated with rotation overnight at 4 °C. Beads were then washed to remove unbound materials. RNA was eluted, isolated, and subjected to qRT-PCR analysis.