Inducible PiggyBac Vector for METTL3 Expression

To generate the inducible PiggyBac donor vector with an amino-terminal FLAG tag fused to METTL3, we first synthesized gBlock (IDT) containing the BamHI-1XFLAG-METTL3-BamHI sequence. This fragment was then cloned into the BamHI entry site in the enhanced piggyBac transposable vector epB-Bsd-TT via cut-ligation to generate the vector, ePB-1xFLAG-METTL3 (Rosa et al. 2014 (link)).
Mettl3 KO mESCs were transfected with an equimolar amount of ePB-1xFLAG-METTL3 and piggyBac transposase (Rosa et al. 2014 (link)). Two days after transfection, cells were plated at clonal density and subjected to a transient puromycin selection (1 µg/mL) for 7 d. Colonies were picked 12 d after transfection, and western blot against endogenous METTL3 (Abcam, ab195352) was performed to identify clones which maintain comparable METTL3 expression between WT mESC cells and the ePB-METTL3 mESC line that was generated from parental Mettl3 KO mESCs. For experiments involving the ePB-METT3 ESC line, all inductions were performed using 100 ng/mL doxycycline (Sigma-Aldrich). A western blot of induced and uninduced cells, probed for METTL3 (Abcam, ab195352) and histone H3 (Abcam, ab1791), is shown in Supplemental Figure 3B. For the m6A-ELISA, mESC lines were cultured in 2i + LIF conditions on gelatin 0.1%-coated plates. Cells were harvested with Accutase (A1110501, Life Technologies).