Next-generation sequencing measuring genome-wide RNA decay by s4U Seq in naïve CD4+ T cells from Mettl3 WT and KO mice

Naïve T cells of Mettl3 KO and WT were pulse labeled by s4U for 15 min before and after IL-7 stimulation for 15min/30min/45min/60min/75min/90min. The total input RNAs or s4U nascent RNAs were isolated and prepared for next-generation sequencing. The RNA response to IL-7 and RNA decay rates were then modeled and calculated, to infer the m6A/Mettl3 effects. Naïve T cells were isolated from Mettl3 KO and WT mice with naïve T cell purification kits (StemCell). The cells were counted and aliquoted 4 million per 1mL FACS buffer per Eppendorf tube. Each tube except t=0 receives 10 µg/ml IL-7 cytokine and incubate in 37℃ incubator. 15 min before spun down and lysed with Trizol, the cells were labeled with 250 µM s4U. The enrichment of s4U-RNA was performed using MTS-biotin chemistry. The input and biotin precipitated RNA were used to prepare library for next-generation sequencing.