Preparation of reduced representation bisulfite sequencing libraries for genome-scale DNA methylation profiling View Full Text


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Article Info

DATE

2011-04

AUTHORS

Hongcang Gu, Zachary D Smith, Christoph Bock, Patrick Boyle, Andreas Gnirke, Alexander Meissner

ABSTRACT

Genome-wide mapping of 5-methylcytosine is of broad interest to many fields of biology and medicine. A variety of methods have been developed, and several have recently been advanced to genome-wide scale using arrays and next-generation sequencing approaches. We have previously reported reduced representation bisulfite sequencing (RRBS), a bisulfite-based protocol that enriches CG-rich parts of the genome, thereby reducing the amount of sequencing required while capturing the majority of promoters and other relevant genomic regions. The approach provides single-nucleotide resolution, is highly sensitive and provides quantitative DNA methylation measurements. This protocol should enable any standard molecular biology laboratory to generate RRBS libraries of high quality. Briefly, purified genomic DNA is digested by the methylation-insensitive restriction enzyme MspI to generate short fragments that contain CpG dinucleotides at the ends. After end-repair, A-tailing and ligation to methylated Illumina adapters, the CpG-rich DNA fragments (40-220 bp) are size selected, subjected to bisulfite conversion, PCR amplified and end sequenced on an Illumina Genome Analyzer. Note that alignment and analysis of RRBS sequencing reads are not covered in this protocol. The extremely low input requirements (10-300 ng), the applicability of the protocol to formalin-fixed and paraffin-embedded samples, and the technique's single-nucleotide resolution extends RRBS to a wide range of biological and clinical samples and research applications. The entire process of RRBS library construction takes ∼9 d. More... »

PAGES

468

References to SciGraph publications

  • 2010-02. Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution in NATURE METHODS
  • 2009-11. Human DNA methylomes at base resolution show widespread epigenomic differences in NATURE
  • 2009-12. BSMAP: whole genome bisulfite sequence MAPping program in BMC BIOINFORMATICS
  • 2008-08. Genome-scale DNA methylation maps of pluripotent and differentiated cells in NATURE
  • 2010-12. BS Seeker: precise mapping for bisulfite sequencing in BMC BIOINFORMATICS
  • 2005-08. Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells in NATURE GENETICS
  • 2008-07. A Bayesian deconvolution strategy for immunoprecipitation-based DNA methylome analysis in NATURE BIOTECHNOLOGY
  • 2006-12. DNA methylation profiling of human chromosomes 6, 20 and 22 in NATURE GENETICS
  • 2010-04. Genome, epigenome and RNA sequences of monozygotic twins discordant for multiple sclerosis in NATURE
  • 2005-09. Methylated-CpG island recovery assay: a new technique for the rapid detection of methylated-CpG islands in cancer in LABORATORY INVESTIGATION
  • 2010-10. Quantitative comparison of genome-wide DNA methylation mapping technologies in NATURE BIOTECHNOLOGY
  • 2008-03. Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning in NATURE
  • 2010-10. Taking the measure of the methylome in NATURE BIOTECHNOLOGY
  • 2007-04. Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome in NATURE GENETICS
  • Journal

    TITLE

    Nature Protocols

    ISSUE

    4

    VOLUME

    6

    Identifiers

    URI

    http://scigraph.springernature.com/pub.10.1038/nprot.2010.190

    DOI

    http://dx.doi.org/10.1038/nprot.2010.190

    DIMENSIONS

    https://app.dimensions.ai/details/publication/pub.1034558482

    PUBMED

    https://www.ncbi.nlm.nih.gov/pubmed/21412275


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