Piercetm magnetic rna protein pull down kit

The in vitro RNA pulldown assay with a biotin-coupled probe was performed by GeneCreate Biotech (Wuhan, China) with Pierce^TM Magnetic RNA-protein Pull-Down Kit (#20164, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Then, the eluted proteins from the sense and antisense groups were used for mass spectrometry analysis.

RNA pulldown assays were performed using the Pierce^TM Magnetic RNA-Protein Pull-Down Kit (#20164, Thermo Fisher Scientific, United States) according to the instructions of the manufacturer. Full-length Gm18840 RNA was obtained using in vitro transcription with the T7 High Yield RNA Transcription Kit (TR101, Vazyme), after which the RNA was biotinylated using the Pierce^TM RNA 3′ End Desthiobiotinylation Kit (# 20163, Thermo Fisher Scientific, United States). Cell extracts were incubated with RNA for 20 min, followed by the addition of nucleic acid-compatible streptavidin magnetic beads (Thermo Fisher Scientific) for further incubation. After washing five times, Gm18840-associated proteins, which were retrieved from beads, were subjected to SDS-PAGE and silver staining. Subsequently, the protein bands were excised and identified by liquid chromatography–mass spectrometry (LC-MS/MS).

RNA was in vitro transcribed with T7 RNA polymerase (Fermentas, EP0111), and was labeled with biotin using Pierce^TM RNA 3ʹ End Desthiobiotinylation Kit (Thermo, 20163) according to the manufacturer’s instructions. In vitro RNA pulldown was performed as previously described^{54 (link)}. Breifly, 50 pmol of labeled RNA in RNA structure buffer (10 mM Tris pH 7, 0.1 M KCl, 10 mM MgCl₂) was heated to 95 °C for 2 min, stand on ice for 3 min, then left at room temperature (RT) for 30 min to allow proper secondary structure formation. Samples were subjected to RNA pulldown using Pierce^TM Magnetic RNA-Protein Pull-Down Kit (Thermo, 20164). Beads were eluted and boiled in 2× SDS protein loading buffer. The recovered proteins were subjected to gradient gel electrophoresis and mass spectrometry (MS) for protein identification or detected by western blot.

A Pierce^TM Magnetic RNA-Protein Pull-Down kit (Thermo Fisher Scientific) was used for RNA pull-down assay. Briefly, the biotin-labeled circ_DOCK1-WT, circ_DOCK1-MUT, and negative control (bio-NC) were generated and interacted with the magnetic beads. HBVSMCs were lysed and incubated with the magnetic beads for 8 h. MiR-409-3p level enriched on the beads was detected by qRT-PCR.
A Magna RIP^TM RNA-Binding Protein Immunoprecipitation kit (Sigma) was used for RNA immunoprecipitation (RIP) analysis. In brief, HBVSMC lysates were incubated with anti-Ago2 or anti-IgG-coated magnetic beads for 6 h. MCL1 and miR-409-3p levels enriched on the beads were measured via qRT-PCR.