Pierce magnetic rna protein pull down kit

Pull-down experiment was performed using the Pierce™ Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific). Briefly, 100 pmol of biotin-labeled miR-1246 or poly-A RNA oligonucleotides (Integrated DNA Technologies) were hybridized to 100 μl streptavidin magnetic beads (Prod#1862766, Thermo Fisher Scientific). The miR-1246-biotin-streptavidin beads were incubated with PANC-1 lysate for 60 min at 4 °C. The lysate-bead mixture was washed three times with washing buffer from the above-mentioned kit. To elute bound proteins, 50 μl of elution buffer was applied, and a magnetic separator was applied to separate the beads from the eluted protein, following the manufacturer’s protocol (Pierce™ Magnetic RNA-Protein Pull-Down Kit, Thermo Fisher scientific). Proteins were separated by SDS-PAGE before mass spectrometry (MS) analysis.

MISSEN full-length sense and antisense sequence was cloned into pEASY-Blunt plasmid and its internal sequences were cloned into pEASY-Blunt plasmid containing the 5′ terminal tRSA tag. The plasmids were used as templates to in vitro transcribe RNA products using the TranscriptAid T7 High Yield Transcription Kit (Thermo, USA). The sense and antisense MISSEN RNAs were further labeled with biotin using Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo, USA). Then the RNA products were purified using the GeneJET RNA Purification Kit (Thermo, USA). 50 pmol RNA per reaction system were denatured 5 min at 85 °C and slowly cooled down to room temperature. The powder of spikelets 3 days after pollination or protoplast cells transfected with HeFP expression vectors were harvested and resuspended with appropriate amount of IP lysis buffer with Rnase inhibitor (Roche, Switzerland) and Cocktail (Thermo, USA). The RNA pulldown assay was performed using the Pierce Magnetic RNA-Protein Pull-Down Kit in accordance with the manufacturer’s instructions (Thermo, USA). The interacted proteins were determined by Mass spectrometry or Western blotting.

The following reagents and instruments were used in this study: dual-luciferase reporter assay system (Promega Madison, US), fetal bovine serum (FBS), Dulbecco's modified eagle medium (DMEM) cell culture medium (Gibco, Rockford, US), radio immunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China), Matrigel (BD, New Jersey, US), cell counting kit-8 (CCK8) assay kit (Dojindo Corp, Kyushu, Japan), Gene Mutation Kit and SYBR Green Premix Ex Taq™ II (TaKaRa, Dalian, China), Pierce^TM Magnetic RNA-Protein Pull-Down kit, Lipofectamine 3000, M-MLV reverse transcriptase kit, miRNA reverse transcriptase kit, TRIzol reagent, and SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific, Inc., Waltham, US). Antibodies were purchased from Santa Cruz (Dallas, US). Propidium iodide and APC-Annexin V were purchased form Sigma-Aldrich (St. Louis, USA). The PsiCHECK™-2 vector was purchased from Promega (Madison, US).