Fig. 1 |. Partial m⁷G TRAC-Seq workflow.

Isolated small RNAs are treated with the bacterial demethylase AlkB to remove the majority of tRNA modifications and then the demethylated small RNAs are treated with NaBH₄ and aniline to induce reduction and cleavage of the RNA backbone at m⁷G-modified sites. Cleavage at the m⁷G results in two products: a 5′ product with a 3′ irregular end that prevents adaptor ligation and therefore cannot be sequenced, and a 3′ product that has both ends available for adaptor ligation. After that a 5′ adaptor is ligated to the cleaved sites of the 3′ cleavage product for the subsequent cDNA library construction. Libraries are sequenced and bioinformatic analysis is performed to identify m⁷G-modified sites.