Library Construction for Genome-wide Bisulfite Sequencing in Plants
拟南芥Bs-seq文库的构建
发布: 2013年12月20日第3卷第24期 DOI: 10.21769/BioProtoc.1013 浏览次数: 14379
评审: Feng Li
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参见作者原研究论文
The authors used this protocol in:
Feb 2013
Abstract
DNA methylation is the most studied epigenetic modification, which involves the addition of a methyl group to the carbon-5 position of cytosine residues in DNA. DNA methylation is important for the regulation of gene expression. Bisulfite sequencing is the gold standard technique for determining genome-wide DNA methylation profiles in eukaryotes. This protocol describes how to prepare libraries of genomic DNA for whole-genome bisulfite sequencing in Arabidopsis, which could be adapted for use in other plant species.
Materials and Reagents
- DNA LoBind 1.5 and 2.0 ml centrifuge tubes (Eppendorf, catalog number: 22431021 and 22431048 , respectively)
- AMPure magnetic beads (Beckman Coulter, catalog number: A63881 )
- Ethanol (70% v/v, ethanol/water)
- TruSeq DNA sample preparation kit (Illumina, catalog number: FC-121-2001 )
- SYBR Safe (Life Technologies, catalog number: S33102 )
- 50 bp ladder (Thermo Fisher Scientific, catalog number: SM0373 )
- Disposable sterile scalpels (Thermo Fisher Scientific catalog number 12-460--452 )
- FalconTM 15 ml conical centrifuge tubes (Thermo Fisher Scientific, catalog number: 14-959-49B )
- QIAquick gel extraction kit (QIAGEN, catalog number: 28704 )
- Qubit dsDNA HS assay kit (Life Technologies, catalog number: Q32851 )
- EpiTect bisulfite kit (QIAGEN, catalog number: 59104 )
- MinElute PCR purification kit (QIAGEN, catalog number: 28004 )
- PfuTurbo Cx hotstart DNA polymerase (Agilent, catalog number: 600410-51 )
- KAPA library quantification kit (Kapa Biosystems, catalog number: KK4824 )
- TE buffer (see Recipes)
- Elution buffer (see Recipes)
Equipment
- S2 adaptive focus acoustic disruptor (Covaris)
- Microtubes with AFA fiber with pre-slit snap-caps (Covaris, catalog number: 520045 )
- Refrigerated water bath circulator (Thermo Fisher Scientific, model: SC100-A10 )
- Thermomixer R (Eppendorf, catalog number: 022670107 )
- MiniSpin PlusTM centrifuge (Eppendorf, catalog number 0 22620100 )
- Magnetic stand (Life Technologies, catalog number 4457858 )
- Vortex mixer (Thermo Fisher Scientific, catalog number 02-215-365 )
- 2100 BioAnalyzer (Agilent, catalog number G2943CA )
- Qubit 2.0 fluorometer (Life Technologies, catalog number Q32866 )
- Horizon 11-14, horizontal gel electrophoresis system (Apogee Designs, model: 11068020 )
- Safe Imager 2.0 blue light transilluminator (Life Technologies, model: G6600 )
- ABI7900HT real-time PCR system (Life Technologies)
- Thermal cycler (C1000 Touch) (Bio-Rad Laboratories, model: 185-1196EDU )
- Pipettors (RAININ, various models)
Procedure
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Amador, D. M., Wang, C., Holland, K. H. and Mou, Z. (2013). Library Construction for Genome-wide Bisulfite Sequencing in Plants. Bio-protocol 3(24): e1013. DOI: 10.21769/BioProtoc.1013.
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分类
植物科学 > 植物分子生物学 > DNA
分子生物学 > DNA > DNA 修饰
系统生物学 > 表观基因组学 > DNA 甲基化
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