S7899

RNA fragmentation is based on the previously described m⁶A-seq protocol [27 (link)] with a few modifications: the total volume of approximately 3 to 5 μg total RNA was adjusted to 18 μl with RNase-free water. The amount of 2 μl of 10X RNA Fragmentation Buffer (100 mM Tris-HCl, 100 mM ZnCl2 in nuclease-free H₂O) was added and incubated in a preheated thermal cycler for approximately 5 to 6 minutes at 70 °C. The reaction was stopped by adding 2 μl of 0.5 M EDTA. Then added to the mixture was 178 μl of H₂O, 20 μl of sodium acetate (3 M [pH 5.2]; S7899; Sigma-Aldrich, St. Louis, MO), 14.4 μl of glycogen (5 mg/mL; AM9510; Thermo Fisher Scientific), and 500 μl of 100% ethanol, and the mixture was incubated at −80 °C overnight. Fragmented RNA was pelleted by centrifuge, washed once with 75% ethanol, and resuspended in ultrapure H₂O (10 μl H₂O per 1 μg human total RNA). The size distribution of fragmented RNA was assessed using High Sensitivity RNA Screentape on TapeStation (5067–5576; Agilent Technologies; Santa Clara, CA). The total RNA was chemically fragmented into approximately 200-nt-long fragments.

Biotinylated (Bio)-TEX41-Wt, Bio-TEX41-Mut and Bio-NC were synthesized firstly. Afterwards, biotin-labeled probes were added into the cell lysates of A375 and SK-MEL-2 to carry out RNA pull down experiment. Subsequently, magnetic beads (HY-K0208, MedChemExpress, NJ, USA) were put into the cell lysates to obtain RNA complex conjoined with magnetic beads. After 2-hour incubation, the beads were washed with buffer solution (S7899, Sigma-Aldrich, St. Louis, MO, USA). The enrichment of miR-103a-3p in RNA-RNA complex was detected by qRT-PCR.

Dasatinib (MedChemExpress, HY-10181) was dissolved in DMSO (75 mg ml^–1, 37.5 mg ml^–1 or 0 mg ml^–1) and 25-fold diluted with 50 mmol per litre sodium acetate buffer (pH 4.6; Sigma, S7899). Mice were treated with dasatinib at a dose of 5 μl per g body weight through oral gavage into the stomach using curved feeding needles (Kent Scientific, FNC-20-1.5-2).