New England Biolabs, E3350S, NEBNext® Enzymatic 5hmC-seq Kit

NEBNext Related Categories Hydroxymethylation Detection and Analysis,, Methylome Analysis,, DNA Methylation Analysis, Specification Materials Required but not Supplied Covaris® instrument and the required tubes or other fragmentation equipment NEBNext Primers for Epigenetics (Unique Dual Index Set 2B) NEB #E3392S (24 reactions) or Set 3 NEB #E3404S (96 reactions) PCR strip tubes or 96-well plates 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 #E3350 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: What is the concentration of the E5hmC-seq™ Adaptor and E5hmC-seq Index Primers? A: The E5hmC-seq Adaptor is 15 µM and the E5hmC-seq Index Primers are 10 µM. @font-face {font-family:"Cambria Math"; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:roman; mso-font-pitch:variable; mso-font-signature:-536870145 1107305727 0 0 415 0;}@font-face {font-family:Calibri; panose-1:2 15 5 2 2 2 4 3 2 4; mso-font-charset:0; mso-generic-font-family:swiss; mso-font-pitch:variable; mso-font-signature:-469750017 -1040178053 9 0 511 0;}p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:""; margin-top:0in; margin-right:0in; margin-bottom:8.0pt; margin-left:0in; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:Calibri; mso-fareast-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}.MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; font-size:11.0pt; mso-ansi-font-size:11.0pt; mso-bidi-font-size:11.0pt; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:Calibri; mso-fareast-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi; mso-font-kerning:0pt; mso-ligatures:none;}.MsoPapDefault {mso-style-type:export-only; margin-bottom:8.0pt; line-height:107%;}div.WordSection1 {page:WordSection1;} Q: Why, at some stages of the E5hmC-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 E5hmC-seq 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-PCR 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 is the expected size of an E5hmC-seq™ library? A: Standard E5hmC-seq libraries have an average size of approximately 500 bp. Library sizes can vary, and 400–600 bp libraries will still work well. Q: How should E5hmC-seq™ libraries be sequenced? A: For Illumina® sequencing, read length is ultimately determined by library size and the user’s requirements. Libraries can be sequenced using 2x76, 2x100 or 2x150 base reads. Q: How should E5hmC-seq™ sequencing data be analyzed? A: In E5hmC-seq libraries, 5hmCs are sequenced as cytosine, whereas 5mC and unmethylated cytosine are sequenced as thymine. As the modified cytosine of interest is not converted and is sequenced as cytosine, established pipelines for EM-seq™ or bisulfite data analysis can be used. We also have a demo pipeline and example data on the GitHub library: https://github.com/nebiolabs/EM-seq/ Q: How deep should E5hmC-seq™ libraries be sequenced? A: The depth of sequencing required is dependent on the user’s application but generally 30X is a suitable starting depth. However, as 5hmC levels can be low and diffuse in certain sample types, it is possible that 100X depth or greater may be required to accurately quantify 5hmC levels. Q: Can the E5hmC-seq™ Adaptor be substituted with another adaptor? A: No. The E5hmC-seq Adaptor has been optimized for use with the NEBNext® Enzymatic 5hmC-seq Kit. Adaptors that are modified to contain 5mC bases are not compatible with this workflow. Q: Can the E5hmC-seq™ Adaptor be used for EM-seq™? A: The E5hmC-seq Adaptor has been optimized for use in the E5hmC-seq workflow. We do not recommend using it to construct EM-seq libraries. Q: Are E5hmC-seq™ libraries directional or non-directional? A: E5hmC-seq libraries are directional. Q: Can other buffers be used in place of the supplied Elution Buffer? A: The Elution Buffer should be used where stated in the protocol, except in the case of long-term storage of PCR-amplified E5hmC-seq™ libraries, where 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) can be used. 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. Q: Do E5hmC-seq™ libraries detect 5mC? A: No, E5hmC-seq libraries specifically detect 5hmC. Furthermore, unmethylated cytosines and 5mC cannot be distinguished. To identify only 5mC, users need to prepare two libraries and then perform a subtractive data analysis. EM-seq™ libraries detect 5mC and 5hmC whereas E5hmC-seq libraries identify 5hmC. Subtracting E5hmC-seq data from EM-seq data therefore provides 5mC information. Q: Are Sample Sheets available for use with the NEBNext® Primers for Epigenetics? A: Sample sheets can be found on the Usage Guidelines section of the NEB #E3404S and #E3392S product pages. Q: Are dual indexed libraries compatible with single end sequencing? A: Yes, the NEBNext Oligos for Illumina, including dual index oligos, are compatible with single read sequencing. Please refer to Illumina’s Indexed Sequencing Overview Guide (current version) for details on dual indexed sequencing workflows on a single-read flow cell or a paired-end flow cell. Q: Does the kit include primers? A: No. The kit can be used with the NEBNext LV Unique Dual Index Primers. For a complete list of low volume (LV) unique dual index primers, please see the third column of NEBNext® Multiplex Oligos Selection Chart. For use with NEBNext library prep kits, consult the library prep kit manual. The NEBNext LV Unique Dual Index Primers manuals also contain tips for setting up ligation reactions. For color balancing options, please see NEBNext® Index Oligo Selector for valid barcode combinations.