M6a antibody

For immunoblotting, primary antibodies specific for YTHDF2 (1:5000, #RN123PW), β-actin (1:5000, #M177-3), Myc (1:5000, #M192-3) were purchased from MBL (MBL, Woburn, MA, USA). NICD antibody (1:3000, #07-1231), HES1 (1:1000, #ab 5702) and HES5 (1:1000, #ab 5708) were obtained from EMD Millipore Corporation (EMD Millipore Corporation, Temecula, CA, USA). Primary antibodies specific for Flag (1:5000, #F7425) were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). GAPDH antibody (1:5000, #AM4300) was obtained from Applied Biological Materials (Applied Biological Materials, Richmond, BC, Canada). FITC (1:500, # 209-095-082) and TRITC (1:500, # 209-025-082) antibodies were purchased from Jackson Immuno Research Laboratories (Jackson Immuno Research Laboratories, West Grove, PA, USA). m⁶A antibody (#A-1801) was purchased from EpiGentek (EpiGentek, Farmingdale, NY, USA).

m6A-seq libraries were prepared with the use of an EpiNext CUT&RUN RNA m6A-Seq Kit (EpiGentek). In brief, total RNA (5 µg) extracted from mouse heart was subjected to immunoprecipitation with the m6A antibody (P-9016, EpiGentek, 1:100 dilution) and cleaved on beads. The beads were then washed, RNA was purified from the beads and subjected to RT, and the resulting cDNA was amplified by PCR. The libraries were sequenced with a NovaSeq 6000 system (Illumina).

Two micrograms of total RNA were deposited on a nylon membrane (FFN10, Beyotime Biotechnology, Shanghai, China), and then the nylon membrane was crosslinked by UV for 3 min. Next, the nylon membrane was stained by using methylene blue. Subsequently, the nylon membrane was blocked for 1 h in blocking buffer, and the membrane was incubated with m6A antibody (1 µg/mL, A-1801, Epigentek Group Inc., Farmingdale, NY) at 4 ℃ overnight. The membrane was incubated with horseradish peroxidase-labeled secondary antibodies at room temperature for 1 h and enhanced chemiluminescent reagent imaging.