Protocol for Use with Standard Insert Libraries (370-420 bp) (NEB#E7120) v2
Authors: England Biolabs New 
Published: July 2024

This protocol details how to construct DNA libraries from start to finish using NEBNext reagents. The corresponding NEB manual is here: https://www.neb.com/-/media/nebus/files/manuals/manuale7120.pdf and this protocol relates to section 1. Overview The Enzymatic Methyl-seq kit (EM-seq) for Illumina contains all the components needed to make libraries that are enzymatically modified to detect 5-methylcytosines (5mC) and 5-hydroxymethylcytosines (5hmC). Figure 1 is an overview of the EM-seq workflow. Firstly, a library is made by ligating EM-seq adaptor to sheared end repaired/dA-tailed genomic DNA. This is followed by two sets of enzymatic conversion steps to differentiate cytosines from 5mC and 5hmC. Finally, libraries are PCR amplified before sequencing. Figure 2. Overview of Sodium Bisulfite Conversion and EM-seq. Figure 2 shows a comparison of the sodium bisulfite and EM-seq methods. Sodium bisulfite treatment of DNA results in the deamination of cytosines into uracils, however the modified forms of cytosine (5mC and 5hmC) are not deaminated. Therefore, the preference of bisulfite to chemically deaminate cytosines enables the methylation status of cytosines to be determined. When bisulfite treated DNA is PCR amplified, uracils are replaced by thymines and the 5mC/5hmC are replaced by cytosines. Once sequenced, unmethylated cytosines are represented by thymines and 5mC and 5hmC are represented by cytosines. By comparing sequences to non-converted genomes the appropriate methylation status can be assessed. Enzymatic Methyl-seq is a two step enzymatic conversion process to detect modified cytosines. The first step uses TET2 and an oxidation enhancer to protect modified cytosines from downstream deamination. TET2 enzymatically oxidizes 5mC and 5hmC through a cascade reaction into 5-carboxycytosine [5-methylcytosine (5mC) -> 5-hydroxymethylcytosine (5hmC) -> 5-formylcytosine (5fC) -> 5-carboxycytosine (5caC)]. This protects 5mC and 5hmC from deamination. 5hmC can also be protected from deamination by glucosylation to form 5ghmc using the oxidation enhancer. The second enzymatic step uses APOBEC to deaminate C but does not convert 5caC and 5ghmC. The resulting converted sequence can be analyzed like bisulfite-treated DNA. Typical aligners used to analyze data include but are not limited to Bismark and BWAMeth. The workflow described in the NEBNext Enzymatic Methyl-seq Kit is user-friendly and enables methylation detection from inputs ranging between 10 ng–200 ng. EM-seq converted DNA is more intact than bisulfite-converted DNA, resulting in libraries with longer sequencing reads, reduced GC bias and more even genome coverage. Please note that the bead volumes provided are sufficient for building standard size libraries described in Section 1, Protocol for use with Standard Size Libraries (370–420 bp). If following the Section 2 Protocol, for use with Large Size Libraries (470–520 bp), users need to supply additional beads due to the increased volumes needed for cleanups. We recommend using SPRIselect‱ Reagent Kit (Beckman Coulter, Inc. #B23317) or AMPure‱ XP Beads (Beckman Coulter, Inc. #A63881). If you are combining leftover beads from E7120 with the above products, we recommend combing with SPRIselect‱ Reagent Kit (Beckman Coulter, Inc. #B23317). Each kit component must pass rigorous quality control standards, and for each new lot the entire set of reagents is functionally validated together by construction of indexed libraries and sequenced on an Illumina sequencing platform. For larger volume requirements, customized and bulk packaging is available by purchasing through the OEM & Custom Solutionsdepartment at NEB. Please contact custom@neb.com for further information.