Anti m6a primary antibody

GenElute mRNA Miniprep Kit (Sigma) was used to extract mRNA from mandibular tissue and ALC cells according to manufacturer’s protocols. At embryonic stages E14.5, mice enamel organ was harvest. To ensure sufficient concentration of mRNA, the enamel organ of 20 biological replicates were pooled for each sample. Dots (50 ng mRNA per 1.5 μl dot) were applied to an Amersham Hybond-N+ membrane (GE Healthcare). After complete drying the mRNA sample, a UV Stratalinker 2400 was used to crosslink mRNA to the membrane by running the autocrosslink program at 3000 kJ. After three times washing in PBST (0.1% Tween-20 in PBS), the membrane was blocked in 5% skim milk in PBST for 2 h. Then, repeating three times PBST wash, the mRNA crosslinked membrane was incubated with primary anti-m6A antibody (212B11, Synaptic Systems) at 1:1000 dilution for 2 h at RT. Repeating three washes in PBST, horseradish peroxidase–conjugated anti-mouse IgG secondary antibody (Jackson ImmunoResearch) was used to incubate for 1 h at RT. Finally, after three washes, the membrane was visualized. Additionally, before incubation with antibodies, the membrane was stained with 0.02% methylene blue in 0.3 M sodium acetate (pH 5.2) to confirm equal mRNA loading. Next, quantified m6A levels were normalized to amount of mRNA loaded. For each time point, three biological samples in technical duplicates were used.

Total RNA was collected from heads or whole flies using standard trizol chloroform extraction. n = 200 head per condition or n = 10 whole flies per condition. PolyA+ RNA was obtained using Dynabeads mRNA Direct Purification Kit (Ambion, 61011). Dots (total RNA or polyA+ RNA) were applied to an Amersham Hybond-N⁺ membrane (GE Healthcare, RPN119B) in duplicate as 100 ng RNA per 1 ul dot. Dots were done on a Dry Membrane in a clean petri dish. The membrane was completely dried before RNA was crosslinked to the membrane using a UV Stratalinker 2400 by running the auto-crosslink program twice. The membrane was then washed in PBST three times 5 min each, blocked with 5% non-fat milk in PBST for 2 h. The membrane was incubated with primary anti-m⁶A antibody (1:1,000, Synaptic Systems, 202003, 2–97) overnight at 4°, then washed in PBST 3 × 5 min, incubated in HRP-conjugated anti-rabbit IgG secondary antibody (1:5000) for 2 h at room temperature, washed in PBST 3 × 5 min, and visualized using ECL prime. The membrane is washed in PBST and then incubated in methylene blue for 15 min, rinsed in PBST and imaged.