RNA bisulfite sequencing of candidate tRNAs in Drosophila melanogaster

Dnmt2 and NSun2 are (cytosine-5) RNA methyltransferases. Using CRISPR/Cas9 we created null mutations in Dnmt2 and NSun2 genes in Drosophila melanogaster. We also ectopically expressed a wild type and catalytically inactive Dnmt2 form in the Dnmt2 mutant background. RNA bisulfite sequencing was used to follow RNA methylation at partical tRNA substrates. Measuring (cytosine-5) RNA methylation levels in various tRNAs from adult wild type controls, Dnmt2 and NSun2 mutants, and ectopically expressed Dnmt2 in the Dnmt2 null mutant background.