Saline sodium citrate solution

A dot blotting assay was performed essentially as previously reported [32 (link)]. Total RNA or poly (A) + mRNA was isolated as described above. Equal amounts of total poly (A) + mRNA samples (2 μg) were denatured at 65°C for 5 min. Then the samples were loaded onto nylon membranes (GE Healthcare, USA) with ice-cold 20× saline sodium citrate solution (Sigma Aldrich) in a dot blot apparatus (Bio-Rad, USA). The membranes were then UV-crosslinked for 5 min, blocked with 5% non-fat milk for 1 hour, incubated with an m⁶A antibody (1:400; ab151230, Abcam) overnight at 4 °C and horseradish peroxidase-conjugated anti-rabbit IgG for 1 hour at room temperature, and finally detected with a 3,3’-diaminobenzidine peroxidase substrate kit. At the same time, the same poly (A) + mRNA (2 μg) samples were spotted onto membranes, UV-crosslinked twice, stained with 0.02% methylene blue in 0.3 M sodium acetate for 2 hours, and washed with ribonuclease-free water for 5 hours, followed by the scanning to indicate the total content of input RNA.

RNA was isolated from various cell lines using RNAiso plus (Takara), and its concentration was adjusted to 50 ng/μl with 36 μl of RNase‐free water. The RNA secondary structure was determined using RNA incubation buffer (mixture of MOPS, formamide, and formaldehyde) and treatment with ice‐cold 20 × saline‐sodium citrate solution (Sigma‐Aldrich), RNA samples (200 ng) were loaded onto an N+ membrane (GE Healthcare) in a dot blot apparatus (Bio‐Rad Laboratories). To guarantee that an equal amount of total RNA was scanned, the RNA was bound to the membrane by UV cross‐linking before being stained with 0.02% methylene blue (Sigma‐Aldrich). Thereafter, the membrane was blocked with skimmed milk, incubated with the m⁶A antibody (1:2000, Synaptic Systems), incubated with the secondary antibody, and finally exposed to the ChemiDoc MP imaging system (Bio‐Rad).