Ab230356

The total RNA content was separated from 1 × 10⁶ cells using the TRIzol reagent (Invitrogen). The RNA m6A level was determined using the m6A RNA Methylation Quantification kit, and the absorbance value was analyzed using the SpectraMax Plus384 microplate Reader (Molecular Device, Sunnyvale CA, USA). Briefly, 150 µg of the total RNA content was dissolved in 500 mL of Rnase-free water and mixed with Biotinylated-Oligo (Promega) at room temperature for 10 min, followed by the addition of Streptavidin-Paramagnetic Particles (Promega). RNA containing polyA⁺ was separated from the solution using magnetic beads with biotin-streptavidin conjugate. The polyA⁺ enriched RNA was fragmented using the RNA Fragmentation Buffer (Millipore, Bedford, MA, USA). Dynabeads containing 5 µg of anti-m6A (at a dilution ratio of 1:1000; ab230356; Abcam) were used to bind to RNA fragments containing m6A methylation prior to the elution of RNA fragments from beads and overnight precipitation at 4°C. The enrichment of m6A in miR-20a-5p or pri-miR-20a-5p was detected using the provided primer-probe sets (Bogu Co., Ltd, Shanghai, China).

RNA co-immunoprecipitation was conducted using the Magna RIP RNA Binding Protein Immunoprecipitation kit (Millipore). Briefly, the cells were lysed, and mixed with anti-m6A (at a dilution ratio of 1:1000, ab230356, Abcam), anti-DiGeorge Critical Region 8 (DGCR8) (at a dilution ratio of 1:1000, ab191875, Abcam) or IgG (at a dilution ratio of 1:2500, ab150077, Abcam). RNA bound to the antibody was pulled down using protein A/G magnetic beads, followed by quantification using real-time qPCR.