New England Biolabs, E3365S, NEBNext® Enzymatic 5hmC-seq Conversion Module

NEBNext Related Categories Hydroxymethylation Detection and Analysis,, Methylome Analysis,, DNA Methylation Analysis, Specification Materials Required but not Supplied If DNA requires fragmentation: NEBNext UltraShear™ (NEB #M7634) or Covaris® instrument and the required tubes or other fragmentation equipment PCR strip tubes or 96-well plates Clean-up beads: SPRIselect™ Reagent Kit (Beckman Coulter®, Inc. #B23317), AMPure® XP beads (Beckman Coulter, Inc. #A63881) or preferred bead manufacturer. Formamide (Sigma #F9037-100 ml), Hi-Di™ Formamide (Thermo Fisher Scientific® #4401457) or 0.1 N NaOH. Formamide is preferred. If using NaOH, please see FAQ on NEB #E3365 FAQ page. 80% Ethanol 1X TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA), low TE (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) or 10 mM Tris-HCl pH 7.5 or 8.0 Nuclease-free Water Magnetic rack/stand, such as NEBNext Magnetic Separation Rack (NEB #S1515) Metal cooling block, such as Diversified Biotech® (#CHAM-1000) PCR machine Agilent® Bioanalyzer®, TapeStation® or other fragment analyzer and associated consumables FAQ Q: What types of samples can be processed using the NEBNext® Enzymatic 5hmC-seq Kit (NEB #E3350) and the NEBNext Enzymatic 5hmC-seq Conversion Module (NEB #E3365)? A: NEBNext Enzymatic 5hmC-seq can be used with genomic DNA, cell-free DNA and FFPE DNA. Q: What are the recommended inputs for the NEBNext® Enzymatic 5hmC-seq Kit (NEB #E3350) and the NEBNext Enzymatic 5hmC-seq Conversion Module (NEB #E3365)? A: The protocol has been optimized for inputs ranging between 0.1–200 ng. Q: What is the difference between the NEBNext® Enzymatic 5hmC-seq Kit (NEB #E3350) and the NEBNext Enzymatic 5hmC-seq Conversion Module (NEB #E3365)? A: The NEBNext Enzymatic 5hmC-seq Kit contains NEBNext Ultra™ II library construction reagents and the E5hmC-seq™ Adaptor, reagents for 5hmC glucosylation and cytosine deamination; NEBNext Q5U® Master Mix and Sample Purification Beads. The Enzymatic 5hmC-seq Conversion Module is supplied with reagents for 5hmC glucosylation and cytosine deamination, and control DNA. It does not contain the E5hmC-seq Adaptor, reagents for library construction and amplification, or Sample Purification Beads. Q: What buffers are recommended for shearing DNA when using the NEBNext Enzymatic 5hmC-seq Kit and the NEBNext 5hmC-seq Conversion Module? A: DNA should be sheared in either 1X TE (10 mM Tris, 1mM EDTA, pH 8.0), low TE (10 mM Tris, 0.1 mM EDTA, pH 8.0) or 10 mM Tris pH 7.5 or 8.0. We do not recommend using 0.1X TE (1 mM Tris, 0.1 mM EDTA, pH 8.0) or water. Q: Why, at some stages of the E5hmC-seq™ Conversion Module 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 E5hmC-seq Conversion Module reaction step. The bead pellet dries quickly after the glucosylation reaction, so it is important to work quickly at this step. In addition, during the post-APOBEC cleanup the beads tend to clump more easily. Do not over-dry the beads before DNA elution as this can result in difficulties in resuspending the beads, and potentially reduced DNA recovery. Q: What levels of conversion are typical with the control DNAs supplied? A: Two DNA controls are provided: unmethylated lambda DNA and 5hmC T4 DNA. 5hmC T4 DNA is hydroxymethylated in the CpG, CHG and CHH contexts. For unmethylated lambda DNA, less than 1% hydroxymethylation is typically observed indicating a conversion efficiency of greater than 99%. For 5hmC T4 DNA (all cytosines are hydroxymethylated), greater than 96% 5hmC is typically detected in all three cytosine sequence contexts. Q: Can enzymatically fragmented DNA be used in E5hmC-seq™? A: We do not recommend using most commercially available enzymatically based DNA fragmentation mixes in the E5hmC-seq workflow, as enzyme-based shearing processes may affect methylation marks on the DNA. However, NEBNext UltraShear™ (NEB #M7634) has been specifically designed to fragment DNA and retain methylation marks. For specific recommendations on pairing NEBNext UltraShear with NEBNext® E5hmC-seq please contact NEB Technical Support. Q: Can I use NaOH (sodium hydroxide) instead of formamide to denature my DNA prior to the deamination reaction step in the E5hmC-seq protocol? A: Yes, it is possible to use NaOH, although formamide is preferred. 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.