5-Hydroxymethylcytosine (5-hmC) Specific Enrichment
5-羟甲基胞嘧啶 (5-hmC)特异性富集
发布: 2012年08月05日第2卷第15期 DOI: 10.21769/BioProtoc.242 浏览次数: 10600
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参见作者原研究论文
The authors used this protocol in:
Jan 2011
Abstract
5-Hydroxymethylcytosine (5-hmC) is a newly discovered DNA modification in mammalian genomes. This protocol is to be 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 specifically, which could in turn be used for high-throughput mapping via next-generation sequencing.
Keywords: 5-hydroxymethylcytocine (5-羟甲基胞嘧啶)
Capture (采集)
Sequencing (测序)
Materials and Reagents
- Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
- Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884 )
- Trizma base (Tris) (Sigma-Aldrich, catalog number: T1503 )
- HEPES (GenScript USA, catalog number: C01621 )
- Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
- Dimethyl sulfoxide (DMSO) (Thermo Fisher Scientific, catalog number: D128 )
- Tween-20 (Sigma-Aldrich, catalog number: P9416 )
- DBCO-S-S-PEG3-Biotin conjugate (Click Chemistry Tools, catalog number: A112P3 )
- 1,4-Dithiothreitol, ultrapure (DTT) (Roche Applied Science, catalog number: 3117006001 )
- Hydroxymethyl Collector (Active Motif, catalog number: 55013 )
- Wizard Genomic DNA Purification Kit (Promega Corporation, catalog number: A1120 )
- 10 kDa Amicon Ultra-0.5 ml Centrifugal Filters (EMD Millipore, catalog number: UFC501008 )
- QIAquick Nucleotide Removal Kit (QIAGEN, catalog number: 28304 )
- Micro Bio-Spin 6 Column (Bio-Rad Laboratories, catalog number: 732-6222 )
- Dynabeads MyOne Streptavidin C1 (Life Technologies, Invitrogen™, catalog number: 650-01 )
- Qiagen MinElute PCR Purification Kit (QIAGEN, catalog number: 28004 )
- UltraPure Agarose (Life Technologies, Invitrogen™, catalog number: 16500500 )
- β-glucosyltransferase (β-GT) (New England Biolabs)
- 10x β-GT reaction buffer (see Recipes)
- 2x binding and washing (B&W) buffer (see Recipes)
- TE (see Recipes)
Equipment
- Sonication device (Covaris)
- Desktop centrifuge
- Water bath (Thermo Fisher Scientific)
- Gel running apparatus (Bio-Rad Corporation)
- Nanodrop 1000 (Thermo Fisher Scientific)
- Labquake tube shaker (Barnstead/Thermolyne)
- Magnetic separation stand (Promega Corporation, catalog number: Z5342 )
- Qubit 2.0 Fluorometer (Life Technologies, Invitrogen™)
Procedure
文章信息
版权信息
© 2012 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Szulwach, K. E., Song, C., He, C. and Jin, P. (2012). 5-Hydroxymethylcytosine (5-hmC) Specific Enrichment. Bio-protocol 2(15): e242. DOI: 10.21769/BioProtoc.242.
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分类
分子生物学 > DNA > DNA 修饰
生物化学 > 糖类 > 葡萄糖
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