New England Biolabs, E7125L, NEBNext® Enzymatic Methyl-seq Conversion Module

NEBNext Related Categories Enzymatic Conversion for DNA Methylation Analysis,, Automation for NEBNext® NGS Library Prep,, DNA Methylation Analysis, Specification Materials Required but not Supplied PCR strip tubes Formamide (Sigma #F9037-100 ml) or 0.1 N NaOH 80% Ethanol 0.1X TE, pH 8.0 Nuclease-free Water Clean-up Beads: NEBNext Sample Purification Beads (supplied with Enzymatic Methyl-seq Kit, NEB #E7120), SPRIselect® Reagent Kit (Beckman Coulter, Inc. #B23317), AMPure® XP Beads (Beckman Coulter, Inc. #A63881) or preferred bead manufacturer. Magnetic rack/stand, such as NEBNext Magnetic Separation Rack (NEB #S1515) PCR machine Bioanalyzer®, TapeStation® and associated consumables or other fragment analyzer FAQ Q: What types of sample can be processed using the NEBNext® Enzymatic Methyl-seq Kit and the Enzymatic Methyl-seq Conversion Module? A: NEBNext Enzymatic Methyl-seq can be used with genomic DNA, cell-free DNA and FFPE DNA. Q: What are the recommended inputs for the NEBNext® Enzymatic Methyl-seq Kit and the Enzymatic Methyl-seq Conversion Module? A: The protocol has been optimized for inputs ranging between 10–200 ng. A modified protocol is also available for inputs up to 500 ng. Q: What is the difference between the NEBNext Enzymatic Methyl-seq Kit and the Enzymatic Methyl-seq Conversion Module? A: The NEBNext Enzymatic Methyl-seq Kit contains all of the components needed to make an EM-seq library, including reagents for 5-mC/5-hmC oxidation and cytosine deamination, library construction reagents and the EM-seq Adaptor, as well as the NEBNext Q5U Master Mix and dual unique primers for library amplification. The Enzymatic Methyl-seq Conversion Module does not contain reagents for library construction and amplification, or adaptors and primers. Q: What buffers are recommended for shearing DNA in NEB’s enzymatic methyl-seq (EM-seq™) workflows? A: For use with the NEBNext® Enzymatic Methyl-seq Kit (NEB #E7120), DNA should be sheared in one of the following buffers: 10 mM Tris-HCl pH 7.5 or pH 8.0, 1X TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA), or low TE (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA). We do not recommend shearing input DNA in 0.1X TE (1 mM Tris-HCl, 0.1 mM EDTA) or water. For use with the NEBNext® Enzymatic Methyl-seq Conversion Module (NEB #E7125), the DNA must not be in a buffer that contains EDTA before proceeding with the Oxidation Reaction. Therefore, it is recommended that the DNA is sheared in 10 mM Tris pH 8.0. Alternatively, if shearing in 1X TE or low TE, a column or bead cleanup followed by elution in 10 mM Tris-HCl pH 8.0 or nuclease-free water is required. Q: Can the “active” TET2 Buffer be stored longer than 4 months? A: No. The “active” TET2 Buffer should be discarded after 4 months. Q: Can the freshly diluted Fe(II) Solution be stored long term? A: No. The freshly diluted iron solution should be discarded immediately after use. Q: Why, at some stages of the EM-seq protocol, do the NEBNext Sample Purification Beads behave differently when cleaning up the sample? A: The behavior of the beads does change depending on the clean-up and the previous EM-seq reaction step. Special care should be taken after APOBEC deamination as the beads tend to clump more easily. Do not over-dry the beads before DNA elution at this step as this can result in difficulties in resuspending the beads, and potentially reduced DNA recovery from the beads. Q: Can enzymatically fragmented DNA be used as input material for EM-seq™? A: NEBNext UltraShear™ is the only enzymatic fragmentation reagent that we recommend upstream of the EM-Seq workflow. We do not recommend other enzyme-based fragmentation methods as they may affect DNA methylation marks. For EM-seq™ use with NEBNext UltraShear™, please follow the protocol in “Section 3” in the linked manual below. https://www.neb.com/-/media/nebus/files/manuals/manualm7634.pdf?rev=64e0e81d781748dba43702e4a78da971&hash=09F5E6499849C9045DFAE1A3AB3BDAA9 Q: Can I use NaOH (sodium hydroxide) instead of formamide to denature my DNA prior to the deamination reaction? A: Yes, it is possible to use NaOH, although formamide is recommended since it is easier to handle. If using NaOH it is critical that the concentration of the NaOH solution is accurate and the volume added to the reaction is exactly 4 μl. NaOH is highly corrosive. Local and institutional safety guidelines must be followed when working with NaOH. If you are making a dilution from 2 N NaOH, ensure the stock solution has at least a pH of 12.5. Verify the pH of the stock solution prior to use. Avoid inaccurate dilutions by making a large enough volume of diluted NaOH to minimize pipetting errors. We recommend making at least 1 ml. Prepare NaOH dilutions fresh or store diluted aliquots at -20°C for up to 6 months to avoid a change in concentration. Solid NaOH is deliquescent (it absorbs water from the atmosphere). It cannot be weighed accurately. Therefore, solutions prepared from NaOH pellets must be titrated to achieve the proper concentration. Q: Is it normal if the Fe(II) solution from the EM-seq product is yellow or a color change is observed? A: Yes, variation in color (colorless to yellow) has been observed. Testing has shown no difference in performance based on this variation. Q: Can I use NaOH (sodium hydroxide) instead of formamide to denature my DNA before the deamination reaction? A: Can I use NaOH (sodium hydroxide) instead of formamide to denature my DNA before the deamination reaction? Yes, it is possible to use NaOH, although formamide is recommended since it is easier to handle. If using NaOH, the concentration of the NaOH solution must be accurate, and the volume added to the reaction is exactly 4 μl. NaOH is highly corrosive. Local and institutional safety guidelines must be followed when working with NaOH. If you are making a dilution from 2 N NaOH, ensure the stock solution has at least a pH of 12.5. Verify the pH of the stock solution before use. Avoid inaccurate dilutions by making a large enough volume of diluted NaOH to minimize pipetting errors. We recommend making at least 1 ml. Prepare NaOH dilutions fresh or store diluted aliquots at -20°C for up to 6 months to avoid a change in concentration. Solid NaOH is deliquescent (it absorbs water from the atmosphere). It cannot be weighed accurately. Therefore, solutions prepared from NaOH pellets must be titrated to achieve the proper concentration. Q: Is it normal if the Fe(II) solution from the EM-seq™ product is yellow or a color change is observed? A: Yes, color variation (colorless to yellow) has been observed. Testing has shown no difference in performance based on this variation.