Direct Bisulfite Sequencing for Examination of DNA Methylation with Gene and Nucleotide Resolution from Brain Tissues

R. Ryley Parrish1, Jeremy J. Day1, Farah D. Lubin1

1 These authors contributed equally to this work.
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 7.24
DOI:  10.1002/0471142301.ns0724s60
Online Posting Date:  July, 2012
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DNA methylation is an epigenetic modification that is essential for the development and mature function of the central nervous system. Due to the relevance of this modification to the transcriptional control of gene expression, it is often necessary to examine changes in DNA methylation patterns with both gene and single‐nucleotide resolution. Here, we describe an in‐depth basic protocol for direct bisulfite sequencing of DNA isolated from brain tissue, which will permit direct assessment of methylation status at individual genes as well as individual cytosine molecules/nucleotides within a genomic region. This method yields analysis of DNA methylation patterns that is robust, accurate, and reproducible, thereby allowing insights into the role of alterations in DNA methylation in brain tissue. Curr. Protoc. Neurosci. 60:7.24.1‐7.24.12. © 2012 by John Wiley & Sons, Inc.

Keywords: bisulfite sequencing; DNA methylation; epigenetics; central nervous system

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Direct Bisulfite Sequencing of DNA from Brain Tissue in Order to Determine DNA Methylation Levels at Gene‐Specific Sites
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Direct Bisulfite Sequencing of DNA from Brain Tissue in Order to Determine DNA Methylation Levels at Gene‐Specific Sites

  • Fresh‐frozen brain tissue (unit 1.1)
  • MicroAmp DNA extraction kit (Qiagen)
  • EpiTect Bisulfite Kit (Qiagen, cat. no. 59104)
  • SYBR Green Supermix (BioRad, cat. no. 170‐8882), or similar master mix
  • Nuclease‐free water
  • Forward and reverse primers to amplify genomic of region of interest
  • ExoSAP‐IT (Affymetrix, cat. no. 78200)
  • Sequencing primers (see annotation to step 9)
  • Standard DNA samples (EpigenDX, cat. no. 80‐8060‐PreMix,
  • PCR tubes
  • Thermal cycler
  • ABI 3730 (or comparable) DNA analyzer
  • Software for analysis of ABI files (see annotation to step 10)
  • Additional reagents and equipment for agarose gel electrophoresis ( appendix 1N) and DNA sequencing (Ausubel et al., , Chapter 7)
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Key References
   Lubin et al., 2008. See above.
  Describes the protocol and uses the direct bisulfite sequencing method to characterize exon‐specific BDNF DNA methylation from brain homogenates.
   Gupta et al., 2010. See above.
  Describes the protocol and its uses with the chromatin immunoprecipitation assay (ChIP) to characterize other epigenetic modifications at DNA methylation sites within gene promoters.
   Miller et al., 2010. See above.
  This paper describes the methylation‐dependent DNA immunoprecipitation (MeDIP) protocol to assess DNA methylation at gene promoter sites.
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