Purebinding rna protein pull down kit

The RNA-binding protein immunoprecipitation (RIP) assay was performed using the RNA Immunoprecipitation Kit (GENESEED, Guangzhou, China). Briefly, CTX-TNA2 cells were lysed by RIP lysis buffer and cell supernatants were incubated overnight at 4°C with primary antibody against Ago2 or normal rat IgG and protein A/G magnetic beads. Then the complex bound to the magnetic bead was eluted. DNA in the complex bound to the magnetic bead was remove by column purification. And the RNA was purified by column purification and analyzed by the RT-qPCR assay to measure XIST and miR-29c-3p expression. The pull-down assay was performed using the PureBinding RNA-Protein pull-down Kit (GENESEED, Guangzhou, China). Briefly, CTX-TNA2 cells were transfected with biotinylated XIST probe (Bio-XIST-probe) or a negative control probe (Bio-NC-probe). At 48 h after transfection, cells were harvested for biotin-based pull-down assay, followed by RT-qPCR assay to determine XIST and miR-29c-3p level.

Biotinylated circCDK13 and its anti-sense sequence were synthesized by GenePharma (Shanghai, China). RNA pull-down assays were performed using a PureBinding® RNA-Protein pull-down Kit (Geneseed, Guangzhou, China) according to the manufacturer’s instructions. Approximately 1 × 10⁷ cells were collected, disrupted, and incubated with 100 μl of streptavidin-coated magnetic beads for 30 min at 4 °C, with rotating at 10 rpm/min, with biotin-labeled circCDK13 probe. The cell lysates were incubated with streptavidin-coated magnetic beads to pull down the biotin-labeled RNA complex. The eluted proteins were separated by SDS-PAGE followed by silver staining (Fast Silver Stain Kit, Beyotime, China). Afterwards, the silver-stained differential protein band in gel was cut off and subjected to digestion with trypsin at 37 °C overnight. The enzyme-digested polypeptide samples were dried and dissolved again for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis (MaxQuant 1.6.17.0.). The specific sequences of the probes can be found in Supplementary Table 4.

An RNA pull-down assay for investigating RNA-protein interactions was performed using a PureBinding^TM RNA-Protein pull-down kit (P0201, GENESEED, Guangdong, China). First, cell samples (1 × 10⁷) were treated with 1 mL of capture buffer mixed with 10 μL of RNase and 10 μL of protease inhibitor. The supernatant was obtained via centrifugation at 14,000 × g for 10 min at 4 °C. To prepare probe-coated beads, a biotinylated circSLC4A7 probe or oligo probe (Gene-Pharma, China) was incubated with 50 μL of streptavidin magnetic beads. Then, 50 pmol probe was added to the beads, and the mixture was incubated at 4 °C with rotation for 30 min. The beads were collected after washing twice with 0.5 mL of capture buffer. Subsequently, the probe-coated beads were mixed with 450 μL of supernatant at 4 °C with rotation for 1 h. Next, the beads were collected and washed with 1 mL of wash buffer mixed with 1 μL of RNase and 1 μL of protease inhibitor 3 times. Finally, the mixture was loaded with 40 μL of capture buffer and 40 μL of loading buffer for subsequent examinations. The complex pulled-down was subjected to SDS-PAGE and analyzed by mass spectrometry.
The sequence of the negative control probe for the RNA pull-down assay was 5′-GCUUAACAUGUAUCUUAUUCGA-3′, and the sequence of the circSLC4A7 probe was 5′-AAUACCUAUAUUUUAGGGCCUU-3′.

Biotin-labeled oligonucleotide probes targeting the junction site of circSERPINE2 and antisense probes were synthesized by RiboBio Co., Ltd. (Guangzhou, China). An RNA pulldown assay was performed with the PureBindingTM RNA–Protein pulldown Kit (Geneseed) according to the manufacturer’s protocol. Briefly, streptavidin-coated magnetic beads were incubated with the biotinylated probes at 4 °C for 1 h. After that, magnetic beads were separated using a magnet and incubated with the eluate from 1.0 × 10⁷ MSCs lysed in lysis buffer for another 2 h at 4 °C. The probe-RNA–protein complex was pulled down and separated using a magnet. The retrieved proteins in the complex were separated from the magnetic beads by boiling for 10 min and then analyzed by western blotting or mass spectrometry (The Medical Research Center of Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China). The probe sequences are shown in Supplementary file1.

RNA pull down was performed to demonstrate the binding between circRNA and miRNA by PureBinding^TM RNA-Protein pull-down Kit (Geneseed, Guangzhou, China). Briefly, chi-circ_0006511 specific probes conjugated with biotin (RiboBio, Guangzhou, China) bind to magnetic beads carrying streptavidin, incubated with cell lysate. Then, the magnetic beads were collected using a magnetic frame, and chi-circ_0006511 was pulled down. The obtained total RNA was reverse transcribed using the Mir-X miRNA First-Strand Synthesis Kit (Takara, Tokyo, Japan) and miRNA was detected by qPCR.

RNA pull-down and mass spectrometry were performed as described previously [51 (link)]. Pure-Binding^TM RNA-Protein pull-down Kit (Geneseed) was purchased for RNA pull-down assay. The bio-labeled RNA and protein complex was used for western blotting and mass spectrometry analysis. The probe for RNA pull-down was listed in Supplementary Table S4.