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Method Article
Described is a two-step labeling process using β-glucosyltransferase (β-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.
5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/α-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by β-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.
1. Genomic DNA Fragmentation
Fragment genomic DNA using sonication to a desired size range suited for the genome-wide sequencing platform. (We usually sonicate to ~300 bp.) Verify the size distribution of the fragmented genomic DNA on 1% agarose gel (Figure 1).
2. DNA Preparation
Determine the starting DNA amounts based on the abundance of 5-hmC in genomic DNA. Since 5-hmC levels vary significantly in different tissue types, starting DNA amounts depend on the 5-hmC levels of the samples. Please refer to Table 1 for examples.
3. β-GT Catalyzed Reaction (Glucose Transfer Reaction)
4. Biotinylation Reaction (Click Chemistry)
5. Capture of 5-hmC-containing DNA
6. Representative Results
If the quality of genomic DNA is high, typical recovery yields after the β-GT and biotinylation reactions are ~60-70%. However, the capture efficiency vary significantly with different tissue types depending on the 5-hmC levels of the samples. Typically, the capture efficiency for brain genomic DNA is ~4-9%, and in some extreme cases, the efficiency may reach up to 12%. For ES cells, the average capture efficiency is ~2-4%, in contrast to ~0.5% for neural stem cells. The lowest efficiency seen so far was for genomic DNA from cancer cells. All enriched DNA is ready for standard next-generation library preparation protocols. In addition, the captured DNA can also be used as template for real-time PCR to detect the enrichment of some fragments compared to the input DNA, if the related primers are available.
Figure 1. Sonicated human genomic DNA fragments in 1% agarose gel. 10 μg of genomic DNA isolated from human iPS cells in 120 μl of 1X TE buffer was sonicated using a sonication device (Covaris). After sonication, 2 μl of the sonicated DNA was loaded onto 1% agarose gel using 100 bp of DNA marker to compare the sizes of the sonicated DNA fragments.
Component | Volume | Final Concentration |
Water | _ μl | |
10 X β-GT Reaction Buffer | 2 μl | 1 X |
Up to 10 μg genomic DNA | _ μl | Up to 500 ng/μl |
UDP-6-N3-Glc (3 mM) | 0.67 μl | 100 μM |
β-GT (40 μM) | 1 μl | 2 μM |
Total volume | 20 μl |
i) For tissue genomic DNA (high 5-hmC content > 0.1%)
Component | Volume | Final Concentration |
Water | _ μl | |
10 X β-GT Reaction Buffer | 10 μl | 1 X |
Up to 20 μg genomic DNA | _ μl | Up to 500 ng/μl |
UDP-6-N3-Glc (3 mM) | 1.33 μl | 100 μM |
β-GT (40 μM) | 2 μl | 2 μM |
Total volume | 40 μl |
ii) For stem cell genomic DNA (median 5-hmC content ~0.05%)
Component | Volume | Final Concentration |
Water | _ μl | |
10 X β-GT Reaction Buffer | 10 μl | 1 X |
Up to 50 μg genomic DNA | _ μl | Up to 500 ng/μl |
UDP-6-N3-Glc (3 mM) | 3.33 μl | 100 μM |
β-GT (40 μM) | 5 μl | 2 μM |
Total volume | 100 μl |
iii) For cancer cell genomic DNA (low 5-hmC content ~0.01%)
Table 1. Examples of amounts of input DNA and labeling reactions using the samples with various 5-hmC levels by the selective chemical labeling method.
Sample | 5-hmC level | Starting DNA (μg) | Recovery after labeling (input to beads) (μg) | Recovery yield | Pull-down DNA (ng) | Pull-down yield |
Adult mouse cerebellum | 0.4% | 10 | 7.5 | 75% | 236 | 3.1% |
Postnatal day 7 mouse cerebellum | 0.1% | 11 | 9 | 82% | 140 | 1.6% |
Mouse ES cell E14 | 0.05% | 60 | 42 | 70% | 350 | 0.8% |
Table 2. Representative results from mouse brain tissues and ES cells.
5-hydroxymethylcytosine (5-hmC) is a recently identified epigenetic modification present in substantial amounts in certain mammalian cell types. The method presented here is for determining the genome-wide distribution of 5-hmC. We use T4 bacteriophage β-glucosyltransferase to transfer an engineered glucose moiety containing an azide group onto the hydroxyl group of 5-hmC. The azide group can be chemically modified with biotin for detection, affinity enrichment, and sequencing of 5-hmC-containing DNA fragments in ma...
No conflicts of interest declared.
This study was supported in part by the National Institutes of Health (GM071440 to C.H. and NS051630/MH076090/MH078972 to PJ).
Name | Company | Catalog Number | Comments |
Reagents | |||
5M Sodium chloride (NaCl) | Promega | V4221 | |
0.5M pH8.0 Ethylenediaminetetraacetic acid (EDTA) | Promega | V4231 | |
1M Trizma base (Tris) pH7.5 | Invitrogen | 15567-027) | |
HEPES 1M, pH7.4 | Invitrogen | 15630 | |
Magnesium chloride (MgCl2) 1M | Ambion | AM9530G | |
Dimethyl sulfoxide (DMSO) | Sigma | D8418 | |
Tween 20 | Fisher BioReagents | BP337-100 | |
DBCO-S-S-PEG3-Biotin conjugate | Click Chemistry Tools | A112P3 | |
1,4-Dithiothreitol, ultrapure (DTT) Superpure | Invitrogen | 15508-013 | |
QIAquick Nucleotide Removal Kit | Qiagen | 28304 | |
Micro Bio-Spin 6 Column | Bio-Rad | 732-6222 | |
Dynabeads MyOne | Invitrogen | 650-01 | |
Streptavidin C1 | |||
Qiagen MinElute PCR Purification Kit | Qiagen | 28004 | |
UltraPure Agarose | Invitrogen | 16500500 | |
UDP-6-N3-glucose | Active Motif | 55013 | |
Enzyme | |||
β-glucosyltransferase (β-GT) | New England Biolab | M0357 | |
Equipment | |||
Sonication device | Covaris | ||
Desktop centrifuge | |||
Water bath | Fisher Scientific | ||
Gel running apparatus | Bio-Rad | ||
NanoDrop1000 | Thermo Scientific | ||
Labquake Tube Shaker | Barnstead | ||
Labquake Tube Shaker | Thermolyne | ||
Magnetic Separation Stand | Promega | Z5342 | |
Qubit 2.0 Fluorometer | Invitrogen | ||
Reagent setup 10 X β-GT Reaction Buffer (500 mM HEPES pH 7.9, 250 mM MgCl2) 2 X Binding and washing (B&W) buffer (10 mM Tris pH 7.5, 1 mM EDTA, 2 M NaCl, 0.02% Tween 20). |
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