Nucleic acid compatible streptavidin magnetic beads

The RNA pull-down assay was performed using the Pierce™ magnetic RNA-protein pull-down kit (Thermo Scientific). Full-length LINC00958 was obtained using in vitro transcription with the T7 high yield RNA transcription kit (TR101; Vazyme). Next, the RNA was biotinylated using the Pierce™ RNA 3′ end desthiobiotinylation kit (Thermo Scientific). Cell extracts were incubated with RNAs for 20 min, followed by incubation with nucleic acid-compatible streptavidin magnetic beads (Thermo Scientific). The samples were washed five times and the LINC00958-associated proteins retrieved from the beads were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and silver staining. The proteins collected from the RNA pull-down were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

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).

The target relationship between miR-370-5p and CHRM3-AS2 was also verified via RNA pull-down assays. Wild-type (cAGGUCAcgucucugcaguuac) or mutant miR-370-5p (cUCCAGUcgucucugcaguuac) were labelled with biotin-conjugated RNA (WT/MT-miR-370-5p) and transfected into U251 cells. After 48 h of transfection, cells were lysed in RIPA reagent and incubated with Nucleic Acid Compatible Streptavidin Magnetic Beads (Thermo Fisher Scientific) at 25°C for 30 min in the dark. The beads were further incubated with Biotin Elution buffer for 30 min, and the eluents were collected for RNA extraction. The expression of CHRM3-AS2 was quantified using qRT-PCR as described earlier.