Amaxa mouse t cell nucleofector kit manual

The WT and catalytic dead Mettl3 genes were cloned into N103 plasmid in Chang lab. To create a catalytic dead mutant Mettl3, we inserted the mutations D395A and W398A (DPPW catalytic motif of METTL3) based on published data, which show that those two residues are absolutely required for METTL3 activity ^{35 (link)–37 (link)}. Mettl3 KO naïve T cells were isolated from Mettl3 KO mice with StemCell Naïve T cell purification kit, then electroporated by nucleofection by exactly following the Amaxa mouse T cell nucleofector kit manual (Lonza). 4 hours after nucleofection in 37°C incubator, the cells were transferred into Rag2^−/− receipt mice by intravenous injection (1 million cells per mice). 4 weeks after transfer, the cells from spleen, periphery lymph nodes, and mesenteric lymph nodes were analyzed by FACS for cell numbers, and naïve Cell marker CD45RB. The CD4+ T cells were isolated from lymph nodes using StemCell CD4+ T cell kit, and total RNA was isolated from the CD4+ T cells using Direct-zol RNA microprep columns (Zymo Research). RT-qPCR was performed using the isolated RNAs. Each group has at least 5 mice to ensure statistical significance.