This study was supported in part by the National Institutes of Health (GM071440 to C.H. and NS051630/MH076090/MH078972 to PJ).

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. &beta;-GT Catalyzed Reaction (Glucose Transfer Reaction)
<ol>
 <li>Mix by pipetting the mixture as detailed in Table 2 and incubate in a 37 &deg;C water bath for 1 hr.</li>
 <li>After incubation, clean up the reaction with QIAquick Nucleotide Removal Kit, using 10 &mu;g of DNA per column. Elute with 30 &mu;l water per column and combine.</li>
</ol>
4. Biotinylation Reaction (Click Chemistry)
<ol>
 <li>Add DBCO-S-S-PEG3-Biotin conjugate working solution (1 mM) in the eluted DNA solution (from step 3) to a final concentration of 150 &mu;M (i.e., 5 &mu;l of working solution per 30 &mu;l DNA solution).</li>
 <li>Mix by pipetting and incubate in a 37 &deg;C water bath for 2 hr.</li>
 <li>Clean up the reaction with QIAquick Nucleotide Removal Kit. The ideal total elution volume is 100 &mu;l.</li>
 <li>Quantify the recovered DNA amount using microliter scale spectrophotometer (e.g., NanoDrop).</li>
</ol>
5. Capture of 5-hmC-containing DNA
<ol>
 <li>Wash 50 &mu;l of Dynabeads MyOne Streptavidin C1 3 times with 1 ml of 1X B&amp;W buffer according to the manufacturer's instructions. Separate the beads with a magnetic stand.</li>
 <li>Add equal volume of 2X B&amp;W buffer to the recovered biotinylated DNA (100 ul) to the washed beads.</li>
 <li>Incubate for 15 min at room temperature with gentle rotation on a rotator.</li>
 <li>Separate the beads with a magnetic stand and wash the beads 3 times with 1 ml of 1X B&amp;W buffer.</li>
 <li>Elute the DNA by incubating the beads in 100 &mu;l of freshly prepared 50 mM DTT for 2 hr at room temperature with gentle rotation on a rotator.</li>
 <li>Separate the beads with a magnetic stand. Aspirate the eluent and load onto a Micro Bio-Spin 6 Column according to the manufacture instruction to remove the DTT. The target DNA is in the solution now.</li>
 <li>Purify the eluted DNA from the previous step by Qiagen MinElute PCR Purification Kit and elute DNA in 10 &mu;l of EB buffer. Quantify DNA using Qubit Fluorometer, or NanoDrop if concentration is higher than 20 ng/ul. The DNA is ready for downstream genome-wide sequencing library preparation.</li>
</ol>
6. Representative Results
If the quality of genomic DNA is high, typical recovery yields after the &beta;-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.
<img alt="Figure 1" src="/files/ftp_upload/4441/4441fig1.jpg" /> 
Figure 1. Sonicated human genomic DNA fragments in 1% agarose gel. 10 &mu;g of genomic DNA isolated from human iPS cells in 120 &mu;l of 1X TE buffer was sonicated using a sonication device (Covaris). After sonication, 2 &mu;l 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.
<table border="1">
 <tbody>
 <tr>
 <td>Component</td>
 <td>Volume</td>
 <td>Final Concentration</td>
 </tr>
 <tr>
 <td>Water</td>
 <td>_ &mu;l</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>10 X &beta;-GT Reaction Buffer</td>
 <td>2 &mu;l</td>
 <td>1 X</td>
 </tr>
 <tr>
 <td>Up to 10 &mu;g genomic DNA</td>
 <td>_ &mu;l</td>
 <td>Up to 500 ng/&mu;l</td>
 </tr>
 <tr>
 <td>UDP-6-N3-Glc (3 mM)</td>
 <td>0.67 &mu;l</td>
 <td>100 &mu;M</td>
 </tr>
 <tr>
 <td>&beta;-GT (40 &mu;M)</td>
 <td>1 &mu;l</td>
 <td>2 &mu;M</td>
 </tr>
 <tr>
 <td>Total volume</td>
 <td>20 &mu;l</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>
i) For tissue genomic DNA (high 5-hmC content &gt; 0.1%)
<table border="1">
 <tbody>
 <tr>
 <td>Component</td>
 <td>Volume</td>
 <td>Final Concentration</td>
 </tr>
 <tr>
 <td>Water</td>
 <td>_ &mu;l</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>10 X &beta;-GT Reaction Buffer</td>
 <td>10 &mu;l</td>
 <td>1 X</td>
 </tr>
 <tr>
 <td>Up to 20 &mu;g genomic DNA</td>
 <td>_ &mu;l</td>
 <td>Up to 500 ng/&mu;l</td>
 </tr>
 <tr>
 <td>UDP-6-N3-Glc (3 mM)</td>
 <td>1.33 &mu;l</td>
 <td>100 &mu;M</td>
 </tr>
 <tr>
 <td>&beta;-GT (40 &mu;M)</td>
 <td>2 &mu;l</td>
 <td>2 &mu;M</td>
 </tr>
 <tr>
 <td>Total volume</td>
 <td>40 &mu;l</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>
ii) For stem cell genomic DNA (median 5-hmC content ~0.05%)
<table border="1">
 <tbody>
 <tr>
 <td>Component</td>
 <td>Volume</td>
 <td>Final Concentration</td>
 </tr>
 <tr>
 <td>Water</td>
 <td>_ &mu;l</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>10 X &beta;-GT Reaction Buffer</td>
 <td>10 &mu;l</td>
 <td>1 X</td>
 </tr>
 <tr>
 <td>Up to 50 &mu;g genomic DNA</td>
 <td>_ &mu;l</td>
 <td>Up to 500 ng/&mu;l</td>
 </tr>
 <tr>
 <td>UDP-6-N3-Glc (3 mM)</td>
 <td>3.33 &mu;l</td>
 <td>100 &mu;M</td>
 </tr>
 <tr>
 <td>&beta;-GT (40 &mu;M)</td>
 <td>5 &mu;l</td>
 <td>2 &mu;M</td>
 </tr>
 <tr>
 <td>Total volume</td>
 <td>100 &mu;l</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>
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.
<table border="1">
 <tbody>
 <tr>
 <td>Sample</td>
 <td>5-hmC level</td>
 <td>Starting DNA (&mu;g)</td>
 <td>Recovery after labeling (input to beads) (&mu;g)</td>
 <td>Recovery yield</td>
 <td>Pull-down DNA (ng)</td>
 <td>Pull-down yield</td>
 </tr>
 <tr>
 <td>Adult mouse cerebellum</td>
 <td>0.4%</td>
 <td>10</td>
 <td>7.5</td>
 <td>75%</td>
 <td>236</td>
 <td>3.1%</td>
 </tr>
 <tr>
 <td>Postnatal day 7 mouse cerebellum</td>
 <td>0.1%</td>
 <td>11</td>
 <td>9</td>
 <td>82%</td>
 <td>140</td>
 <td>1.6%</td>
 </tr>
 <tr>
 <td>Mouse ES cell E14</td>
 <td>0.05%</td>
 <td>60</td>
 <td>42</td>
 <td>70%</td>
 <td>350</td>
 <td>0.8%</td>
 </tr>
 </tbody>
</table>
Table 2. Representative results from mouse brain tissues and ES cells.

<ol>
	<li>Jaenisch, R., Bird, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+regulation+of+gene+expression:+how+the+genome+integrates+intrinsic+and+environmental+signals.">Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals.</a> Nat. Genet. , 245-254 (2003).</li><li>Ito, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Tet+proteins+in+5mC+to+5hmC+conversion,+ES-cell+self-renewal+and+inner+cell+mass+specification.">Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification.</a> Nature. 466, 1129-1133 (2010).</li><li>Tahiliani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+5-methylcytosine+to+5-hydroxymethylcytosine+in+mammalian+DNA+by+MLL+partner+TET1.">Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1.</a> Science. 324, 930-935 (2009).</li><li>Kriaucionis, S., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nuclear+DNA+base+5-hydroxymethylcytosine+is+present+in+Purkinje+neurons+and+the+brain.">The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain.</a> Science. 324, 929-930 (2009).</li><li>Ko, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+hydroxylation+of+5-methylcytosine+in+myeloid+cancers+with+mutant+TET2.">Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2.</a> Nature. 468, 839-843 (2010).</li><li>Koh, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tet1+and+tet2+regulate+5-hydroxymethylcytosine+production+and+cell+lineage+specification+in+mouse+embryonic+stem+cells.">Tet1 and tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells.</a> Cell Stem Cell. 8, 200-213 (2011).</li><li>Iqbal, K., Jin, S. G., Pfeifer, G. P., Szabo, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+the+paternal+genome+upon+fertilization+involves+genome-wide+oxidation+of+5-methylcytosine.">Reprogramming of the paternal genome upon fertilization involves genome-wide oxidation of 5-methylcytosine.</a> Proceedings of the National Academy of Sciences of the United States of America. 108, 3642-3647 (2011).</li><li>Wossidlo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-Hydroxymethylcytosine+in+the+mammalian+zygote+is+linked+with+epigenetic+reprogramming.">5-Hydroxymethylcytosine in the mammalian zygote is linked with epigenetic reprogramming.</a> Nat. Commun. 2, 241 (2011).</li><li>Gu, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+Tet3+DNA+dioxygenase+in+epigenetic+reprogramming+by+oocytes.">The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes.</a> Nature. 477, 606-610 (2011).</li><li>Dawlaty, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tet1+is+dispensable+for+maintaining+pluripotency+and+its+loss+is+compatible+with+embryonic+and+postnatal+development.">Tet1 is dispensable for maintaining pluripotency and its loss is compatible with embryonic and postnatal development.</a> Cell Stem Cell. 9, 166-175 (2011).</li><li>Huang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+behaviour+of+5-hydroxymethylcytosine+in+bisulfite+sequencing.">The behaviour of 5-hydroxymethylcytosine in bisulfite sequencing.</a> PLoS One. 5, e8888 (2010).</li><li>Song, C. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+chemical+labeling+reveals+the+genome-wide+distribution+of+5-hydroxymethylcytosine.">Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine.</a> Nat. Biotechnol. 29, 68-72 (2011).</li><li>Pastor, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+mapping+of+5-hydroxymethylcytosine+in+embryonic+stem+cells.">Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells.</a> Nature. 473, 394-397 (2011).</li><li>Matarese, F., Pau, C. a. r. r. i. l. l. o. -. d. e. S. a. n. t. a., E, ., Stunnenberg, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-Hydroxymethylcytosine:+a+new+kid+on+the+epigenetic+block.">5-Hydroxymethylcytosine: a new kid on the epigenetic block.</a> Mol. Syst. Biol. 7, 562 (2011).</li><li>Szwagierczak, A., Bultmann, S., Schmidt, C. S., Spada, F., Leonhardt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+enzymatic+quantification+of+5-hydroxymethylcytosine+in+genomic+DNA.">Sensitive enzymatic quantification of 5-hydroxymethylcytosine in genomic DNA.</a> Nucleic Acids Res. 38, 181 (2010).</li><li>Terragni, J., Bitinaite, J., Zheng, Y., Pradhan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+characterization+of+recombinant+&#946;-glucosyltransferase+and+analysis+of+global+5-hydroxymethylcytosine+in+unique+genomes.">Biochemical characterization of recombinant &#946;-glucosyltransferase and analysis of global 5-hydroxymethylcytosine in unique genomes.</a> Biochemistry. , (2012).</li><li>Rusmintratip, V., Sowers, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+unexpectedly+high+excision+capacity+for+mispaired+5-hydroxymethyluracil+in+human+cell+extracts.">An unexpectedly high excision capacity for mispaired 5-hydroxymethyluracil in human cell extracts.</a> Proc. Natl. Acad. Sci. U.S.A. 97, 14183-14187 (2000).</li><li>Globisch, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+distribution+of+5-hydroxymethylcytosine+and+search+for+active+demethylation+intermediates.">Tissue distribution of 5-hydroxymethylcytosine and search for active demethylation intermediates.</a> PLoS One. 5, e15367 (2010).</li><li>Yildirim, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mbd3/NURD+Complex+Regulates+Expression+of+5-Hydroxymethylcytosine+Marked+Genes+in+Embryonic+Stem+Cells.">Mbd3/NURD Complex Regulates Expression of 5-Hydroxymethylcytosine Marked Genes in Embryonic Stem Cells.</a> Cell. 147, 1498-1510 (2011).</li><li>Szulwach, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+5-hydroxymethylcytosine+into+the+epigenomic+landscape+of+human+embryonic+stem+cells.">Integrating 5-hydroxymethylcytosine into the epigenomic landscape of human embryonic stem cells.</a> PLoS Genet. 7, e1002154 (2011).</li><li>Szulwach, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-hmC-mediated+epigenetic+dynamics+during+postnatal+neurodevelopment+and+aging.">5-hmC-mediated epigenetic dynamics during postnatal neurodevelopment and aging.</a> Nat. Neurosci. 14, 1607-1616 (2011).</li></ol>

No conflicts of interest declared.

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 &beta;-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 mammalian genomes. This protocol has advantages over 5-hmC antibody-based hydroxymethylated DNA immunoprecipitation (hMeDIP-Seq)13-15, although the hMeDIP is simple, it has a strong bias towards high-density 5-hmC regions and usually gives inconsistent results from independent pull-downs. Our method would not introduce such bias.
There are, however, two possible concerns in terms of our labeling and capture method. The first concern could be the specificity of the labeling and capture. Since a recent report indicated that 5-hydroxymethyluracil (5-hmU) can also serve as a substrate of &beta;-GT, false-positive signals might be introduced, leading to enriched fragments that are nonspecific to 5-hmC-relatedness16. This concern might be unfounded, though, since highly active 5-hydroxymethyluracil-DNA glycosylases are constantly removing this DNA damage, resulting in essentially undetectable 5-hmU in mammalian genome17,18. The second concern is the efficiency of the labeling and the capture. Since 5-hmC levels vary significantly in different tissues and cells, ranging from 0.5% in brain tissues to 0.05% in mES cells and 0.01% in cancer cells, it is essential that our methods be applicable to all these tissues in terms of the downstream applications of the labeled and captured genomic DNA. Our experience shows that the methods described here are indeed sensitive and specific enough to analyze all these tissues for the purpose of either quantification-based comparison of 5-hmC levels among the tissues or for downstream applications, such as deep sequencing to map the distribution of the 5-hmC in the genome19-21.
The key to our method is the use of an appropriate amount of starting genomic DNA. If the genomic DNA concentration is too low, the labelling efficiency and specificity will decrease accordingly. Based on our experience, even though the abundance of 5-hmC is high enough in DNA from brain tissues, if the concentration is lower than 25 ng/&mu;l in 20 &mu;l of reaction, the labelling and capture efficiency, as well as the specificity, will be significantly reduced. For the low-abundance DNA samples, in addition to the concentration requirement, the total amount of DNA is essential to get high and specific capture efficiency. Thus, although one needs only 25 ng of 5-hmC-enriched DNA fragments for the downstream standard library generation protocols employed by next-generation sequencing platforms, the starting amount of genomic DNA from brain tissues should not be less than 2 &mu;g (and the ideal starting amount is 5-10 &mu;g). Through trial and error, we have optimized the starting amount of genomic DNA from different tissues with variable 5-hmC abundances to get high labeling efficiency and specificity, as detailed in Table 2.
In summary, our method is qualified to precisely label genomic 5-hmC and specifically capture the 5-hmC-containing fragments, making it a successful protocol in terms of sensitivity and specificity for downstream next-generation sequencing assays.

<table border="1">
<tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalog #</td>
 <td>Comment</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagents</td>
 </tr>
 <tr>
 <td>5M Sodium chloride (NaCl)</td>
 <td>Promega</td>
 <td>V4221</td>
 <td></td>
 </tr>
 <tr>
 <td>0.5M pH8.0 Ethylenediaminetetraacetic acid (EDTA)</td>
 <td>Promega</td>
 <td>V4231</td>
 <td></td>
 </tr>
 <tr>
 <td>1M Trizma base (Tris) pH7.5</td>
 <td>Invitrogen</td>
 <td>15567-027)</td>
 <td></td>
 </tr>
 <tr>
 <td>HEPES 1M, pH7.4</td>
 <td>Invitrogen</td>
 <td>15630</td>
 <td></td>
 </tr>
 <tr>
 <td>Magnesium chloride (MgCl2) 1M</td>
 <td>Ambion</td>
 <td>AM9530G</td>
 <td></td>
 </tr>
 <tr>
 <td>Dimethyl sulfoxide (DMSO)</td>
 <td>Sigma</td>
 <td>D8418</td>
 <td></td>
 </tr>
 <tr>
 <td>Tween 20</td>
 <td>Fisher BioReagents</td>
 <td>BP337-100</td>
 <td></td>
 </tr>
 <tr>
 <td>DBCO-S-S-PEG3-Biotin conjugate</td>
 <td>Click Chemistry Tools</td>
 <td>A112P3</td>
 <td></td>
 </tr>
 <tr>
 <td>1,4-Dithiothreitol, ultrapure (DTT) Superpure</td>
 <td>Invitrogen</td>
 <td>15508-013</td>
 <td></td>
 </tr>
 <tr>
 <td>QIAquick Nucleotide Removal Kit</td>
 <td>Qiagen</td>
 <td>28304</td>
 <td></td>
 </tr>
 <tr>
 <td>Micro Bio-Spin 6 Column</td>
 <td>Bio-Rad</td>
 <td>732-6222</td>
 <td></td>
 </tr>
 <tr>
 <td>Dynabeads MyOne </td>
 <td>Invitrogen</td>
 <td>650-01</td>
 <td></td>
 </tr>
 <tr>
 <td>Streptavidin C1</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Qiagen MinElute PCR Purification Kit</td>
 <td>Qiagen</td>
 <td>28004</td>
 <td></td>
 </tr>
 <tr>
 <td>UltraPure Agarose</td>
 <td>Invitrogen</td>
 <td>16500500</td>
 <td></td>
 </tr>
 <tr>
 <td>UDP-6-N3-glucose</td>
 <td>Active Motif</td>
 <td>55013</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Enzyme</td>
 </tr>
 <tr>
 <td>&beta;-glucosyltransferase (&beta;-GT)</td>
 <td>New England Biolab</td>
 <td>M0357</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Sonication device</td>
 <td>Covaris</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Desktop centrifuge</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Water bath</td>
 <td>Fisher Scientific</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Gel running apparatus</td>
 <td>Bio-Rad</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>NanoDrop1000</td>
 <td>Thermo Scientific</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Labquake Tube Shaker</td>
 <td>Barnstead</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Labquake Tube Shaker</td>
 <td>Thermolyne</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Magnetic Separation Stand</td>
 <td>Promega</td>
 <td>Z5342</td>
 <td></td>
 </tr>
 <tr>
 <td>Qubit 2.0 Fluorometer</td>
 <td>Invitrogen</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent setup 
10 X &beta;-GT Reaction Buffer (500 mM HEPES pH 7.9, 250 mM MgCl2) 2 X Binding and washing (B&amp;W) buffer (10 mM Tris pH 7.5, 1 mM EDTA, 2 M NaCl, 0.02% Tween 20).</td>
 </tr>
 </tbody>
</table>

selective capture of 5-hydroxymethylcytosine from genomic dna

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)/&alpha;-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 &beta;-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.

Watch this Scientific Journal Video about Selective Capture of 5-hydroxymethylcytosine from Genomic DNA at JoVE.com

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-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 ...

selective-capture-of-5-hydroxymethylcytosine-from-genomic-dna

Nanyang Technological University

Research

JoVE Journal

Biology

11.9K Views.  Emory University School of Medicine. 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.

Video: Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

Isolation of Genomic DNA from Mouse Tails

Large Insert Environmental Genomic Library Production

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE <a href="http://www.epibio.com/item.asp?ID=385">http://www.epibio.com/item.asp?ID=385</a>). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.

Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system.

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent ...

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ancient bones. The method greatly reduces sample destruction and sequencing demands relative to direct PCR or shotgun sequencing approaches. We used this method to reconstruct the complete mitochondrial DNA (mtDNA) genomes of five Neandertals from across their geographic range. The mtDNA genetic diversity of the late Neandertals was approximately three times lower than that of contemporary modern humans. Together with analyses of mtDNA protein evolution, these data suggest that the long-term effective population size of Neandertals was smaller than that of modern humans and extant great apes.

We present a method of targeted ancient DNA sequence retrieval, which we used to reconstruct the complete mitochondrial genomes of five Neandertal individuals. Comparison of these sequences with present day humans suggests that Neandertals had a long term low effective population size.

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ...

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be produced to contain any desired DNA or RNA sequence and can be prepared to include a wide variety of base and sugar modifications. Moreover, oligos can be designed to mimic specific nucleic acid alterations and thus, can serve as important tools to investigate effects of DNA damage and mechanisms of repair. We found that Thermo Scientific Dharmacon RNA-containing oligos with a length between 50 and 80 nucleotides can be particularly suitable to study, in vivo, functions and consequences of chromosomal RNA/DNA hybrids and of ribonucleotides embedded into DNA. RNA/DNA hybrids can readily form during DNA replication, repair and transcription, however, very little is known about the stability of RNA/DNA hybrids in cells and to which extent these hybrids can affect the genetic integrity of cells. RNA-containing oligos, therefore, represent a perfect vector to introduce ribonucleotides into chromosomal DNA and generate RNA/DNA hybrids of chosen length and base composition. Here we present the protocol for the incorporation of ribonucleotides into the genome of the eukaryotic model system yeast /Saccharomyces cerevisiae/. Yet, our lab has utilized Thermo Scientific Dharmacon RNA-containing oligos to generate RNA/DNA hybrids at the chromosomal level in different cell systems, from bacteria to human cells.

This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high purity DNA. Based on multi-well filtration for bacterial lysate clearance and DNA purification, this method creates a streamlined process for plasmid preparation. Filter plates containing silica-based media can easily be processed by vacuum filtration or centrifuge to yield appreciable quantities of plasmid DNA. Quantitative analyses determine the purified plasmid DNA is consistently of high quality with average OD260/280 ratios of 1.97. Overall, plasmid yields offer more pure DNA for downstream applications, such as sequencing and cloning. This streamlined method of using AcroPrep Advance Filter Plates allows for manual, semi-automated or fully-automated processing.

This protocol is a cost effective alternative for efficient parallel clarification and plasmid DNA purification from E. coli cultures. The AcroPrep Advance process starts with an optimized lysate clarification filter plate followed by purification on a high binding capacity DNA binding filter plate.

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high ...

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova &amp; Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).
Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

We present a public computational web site for the analysis of genomic sequences. It detects DNA sequence patterns with various non-random nucleotide compositions. This resource also generates randomized sequences with diverse levels of complexity.

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Extraction of High Molecular Weight DNA from Microbial Mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA2,3 during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes4,5,6.
The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 &mu;g of HMW DNA (35-50 kb) per gram of mat sample, with an A260/280 ratio of 1.6. Furthermore, amplification of 16S rRNA genes7 suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.

We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction  of high-quality, high molecular weight ...

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any two individuals is greater than previously thought3. In contrast, array-based genotyping has failed to identify a significant contribution of common sequence variants to the phenotypic variability of common disease4,5. Taken together, these observations have led to the evolution of the Common Disease / Rare Variant hypothesis suggesting that the majority of the "missing heritability" in common and complex phenotypes is instead due to an individual's personal profile of rare or private DNA variants6-8. However, characterizing how rare variation impacts complex phenotypes requires the analysis of many affected individuals at many genomic loci, and is ideally compared to a similar survey in an unaffected cohort. Despite the sequencing power offered by today's platforms, a population-based survey of many genomic loci and the subsequent computational analysis required remains prohibitive for many investigators.
To address this need, we have developed a pooled sequencing approach1,9 and a novel software package1 for highly accurate rare variant detection from the resulting data. The ability to pool genomes from entire populations of affected individuals and survey the degree of genetic variation at multiple targeted regions in a single sequencing library provides excellent cost and time savings to traditional single-sample sequencing methodology. With a mean sequencing coverage per allele of 25-fold, our custom algorithm, SPLINTER, uses an internal variant calling control strategy to call insertions, deletions and substitutions up to four base pairs in length with high sensitivity and specificity from pools of up to 1 mutant allele in 500 individuals. Here we describe the method for preparing the pooled sequencing library followed by step-by-step instructions on how to use the SPLINTER package for pooled sequencing analysis (<a href="http://www.ibridgenetwork.org/wustl/splinter" target="_blank">http://www.ibridgenetwork.org/wustl/splinter</a>). We show a comparison between pooled sequencing of 947 individuals, all of whom also underwent genome-wide array, at over 20kb of sequencing per person. Concordance between genotyping of tagged and novel variants called in the pooled sample were excellent. This method can be easily scaled up to any number of genomic loci and any number of individuals. By incorporating the internal positive and negative amplicon controls at ratios that mimic the population under study, the algorithm can be calibrated for optimal performance. This strategy can also be modified for use with hybridization capture or individual-specific barcodes and can be applied to the sequencing of naturally heterogeneous samples, such as tumor DNA.

Pooled DNA sequencing is a fast and cost-effective strategy to detect rare variants associated with complex phenotypes in large cohorts. Here we describe the computational analysis of pooled, next-generation sequencing of 32 cancer-related genes using the SPLINTER software package. This method is scalable, and applicable to any phenotype of interest.

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any ...