5-Hydroxymethylcytosine (5-hmC) Specific Enrichment

Materials and Reagents

1. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number:  S3014 )
2. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number:  E9884 )
3. Trizma base (Tris) (Sigma-Aldrich, catalog number:  T1503 )
4. HEPES (GenScript USA, catalog number:  C01621 )
5. Magnesium chloride (MgCl₂) (Sigma-Aldrich, catalog number:  M8266 )
6. Dimethyl sulfoxide (DMSO) (Thermo Fisher Scientific, catalog number:  D128 )
7. Tween-20 (Sigma-Aldrich, catalog number:  P9416 )
8. DBCO-S-S-PEG3-Biotin conjugate (Click Chemistry Tools, catalog number:  A112P3 )
9. 1,4-Dithiothreitol, ultrapure (DTT) (Roche Applied Science, catalog number: 3117006001 )
10. Hydroxymethyl Collector (Active Motif, catalog number: 55013 )
11. Wizard Genomic DNA Purification Kit (Promega Corporation, catalog number:  A1120 )
12. 10 kDa Amicon Ultra-0.5 ml Centrifugal Filters (EMD Millipore, catalog number:  UFC501008 )
13. QIAquick Nucleotide Removal Kit (QIAGEN, catalog number: 28304 )
14. Micro Bio-Spin 6 Column (Bio-Rad Laboratories, catalog number:  732-6222 )
15. Dynabeads MyOne Streptavidin C1 (Life Technologies, Invitrogen™, catalog number:  650-01 )
16. Qiagen MinElute PCR Purification Kit (QIAGEN, catalog number: 28004 )
17. UltraPure Agarose (Life Technologies, Invitrogen™, catalog number:  16500500 )
18. β-glucosyltransferase (β-GT) (New England Biolabs)
19. 10x β-GT reaction buffer (see Recipes)
20. 2x binding and washing (B&W) buffer (see Recipes)
21. TE (see Recipes)
    

Equipment

1. Sonication device (Covaris)
2. Desktop centrifuge
3. Water bath (Thermo Fisher Scientific)
4. Gel running apparatus (Bio-Rad Corporation)
5. Nanodrop 1000 (Thermo Fisher Scientific)
6. Labquake tube shaker (Barnstead/Thermolyne)
7. Magnetic separation stand (Promega Corporation, catalog number:  Z5342 )
8. Qubit 2.0 Fluorometer (Life Technologies, Invitrogen™)

Procedure