Epiquik fluorometric m6a rna methylation quantification kit

mRNA methylation levels were measured as previously described (Park, Sporer, et al. 2023 ). SK1 cultures were sporulated for four hours. Cells were lysed using the FastPrep (MP) as described above. Total RNA was then harvested using the Qiagen RNeasy kit with on-column DNase treatment. mRNA was purified from these total RNA samples using Oligod(T) magnetic beads (Thermofisher). mRNA methylation was measured using the EpiQuik fluorometric m⁶A RNA methylation quantification kit (EpiGenTek). The kit protocol was followed as written. Briefly, purified mRNA was bound to wells and then washed before the addition of an antibody to m⁶A. Wells were then washed again before the addition of a detection antibody followed by an enhancer/developer solution. The fluorescence was measured using a Synergy plate reader (BioTek) at 530_EX/590_EM nm and m⁶A levels were quantified for each sample using a standard curve generated from a positive control provided with the kit. Measurements from an ime4Δ/Δ strain were used to subtract background from the data collected from the vir1Δ/Δ mutant.

SK1 cultures were sporulated for four hours as described above. Cells were lysed using the FastPrep (MP) with four rounds of bead beating at 4.0 m/s for 40 seconds with one minute on ice in between each round. Total RNA was then harvested using the Qiagen RNeasy kit. mRNA was purified from these total RNA samples using Oligod(T) magnetic beads (Thermofisher). mRNA methylation was measured using the EpiQuik fluorometric m⁶A RNA methylation quantification kit (EpiGenTek). Briefly, purified mRNA was bound to wells and then washed before the addition of an antibody to m⁶A. Wells were then washed again before the addition of a detection antibody followed by an enhancer/developer solution. The fluorescence was measured using a plate reader at 530_EX/590_EM nm and m⁶A levels were quantified for each sample using a standard curve generated from a positive control provided with the kit. Measurements from an ime4Δ/Δ strain were used to subtract background from the data collected from other mutants.