RNA half-life measurement

Mettl3-CKO and control mice were sacrificed at p1, and the retinas were collected and soaked in DMEM supplemented with actinomycin D (5 μm, Sigma) to inhibit transcription and cultured in a CO₂ incubator at 37℃. The retinas were collected directly into TRIzoL at 0 hr, 4 hr, 8 hr, and 16 hr after drug treatment. Total RNA was extracted according to the manufacturer’s instructions. Two micrograms of total RNA from each sample was reverse transcribed into cDNA using SuperScript II (Thermo Fisher) and subjected to qPCR measurements using SYBR Green mix (Roche) and LightCycler LC480 (Roche). The primers are listed in the key resources table. The relative mRNA concentrations at each time point against time point 0 were calculated, the ln^{mRNA concentration} at time points 0, 4, 8, and 16 hr were plotted to perform a linear regression analysis as a function of time, and the slope of the line was identified as the decay rate (k). The half-life was calculated with the following formula: t (1/2) = ln2/k (Chen et al., 2008).