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  • FAQ: Is library prep workflow different when NEBNext Methylated Adaptor is used?

    If the libraries are for whole genome sequencing or targeted resequencing, there is no change in library prep workflow when NEBNext Methylated Adaptor is used. However, if the libraries are for bisulfite sequencing, there are two important changes to the workflow: 1) A bisulfite treatment step is required after adaptor ligation/USER cleavage and before PCR amplification. There are many bisulfite conversion kits commercially available, including EpiMark Bisulfite Conversion Kit from New England Biolabs (NEB #E3318); and 2) NEBNext High-Fidelity 2X PCR Master Mix and other high fidelity DNA polymerases including Phusion DNA polymerase should not be used to amplify bisulfite converted, uracil-containing DNA libraries. Instead, the bisulfite converted DNA should be PCR-amplified using a DNA polymerase that can bypass uracil, for example, EpiMark Hot Start Taq DNA Polymerase from New England Biolabs (NEB #M0490).