The experiment concept and in vitro validation of the recombinant protein pA-M.EcoGII in BIND&MODIFY . A The experiment outlines of BIND&MODIFY. After light fixation and permeabilization, the cells were tethered to Concanavalin A magnetic beads (which bind specifically to mannosyl- and glucosyl-containing extracellular glycoproteins on the cell membrane) for purification in the following steps. Step1 Antibody binding; Step 2 pA-M.EcoGII binding; Step 3 Methylation activation; Step 4 ONT sequencing. Firstly, recombinant protein pA-M.EcoGII was tethered to desired sites under antibody guidance. Then the M.EcoGII m6A methylation activity was locally activated with the addition of S-adenosylmethionine to modify the nearby regions to label the genomic DNA with targeted binding proteins. Finally genomic DNA was extracted to prepare the library for ONT nanopore sequencing. After sequencing, the data was processed as genome alignment and m6A base calling (refer to detail protocol).
