A Detailed Protocol for Formaldehyde‐Assisted Isolation of Regulatory Elements (FAIRE)

Jeremy M. Simon1, Paul G. Giresi1, Ian J. Davis2, Jason D. Lieb2

1 Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 2 Carolina Center for Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 21.26
DOI:  10.1002/0471142727.mb2126s102
Online Posting Date:  April, 2013
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Abstract

Nucleosome displacement is a key event in the regulation of gene expression in the eukaryotic genome. This unit details an approach called Formaldehyde‐Assisted Isolation of Regulatory Elements (FAIRE) for isolating nucleosome‐depleted regions. FAIRE does not rely on the use of antibodies or enzymes, and has proven successful in most eukaryotic cells and tissues. The set of regulatory elements enriched by FAIRE is similar to those identified through DNase hypersensitivity. The enriched fragments can be detected by quantitative PCR, tiling DNA microarrays, or next‐generation sequencing. Although the signal‐to‐noise ratio is typically lower than that observed for DNase assays, FAIRE has high sample‐to‐sample reproducibility, requires very low amounts of input material, is inexpensive, is amenable to high‐throughput adaptations, and is a relatively simple procedure with a high rate of success, even for those without extensive experience in molecular biology protocols. Curr. Protoc. Mol. Biol. 102:21.26.1–21.26.15. © 2013 by John Wiley & Sons, Inc.

Keywords: chromatin; regulatory elements; next‐generation sequencing; formaldehyde; DNase; ChIP

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Sample Preparation for Performing FAIRE on Adherent or Suspension Cells Without Large Fat Deposits
  • Support Protocol 1: Sample Preparation for Performing FAIRE on Adherent Lipid‐Laden Cells (or if Bead‐Beater is Unavailable)
  • Support Protocol 2: Sample Preparation for Performing FAIRE on Frozen Tissues
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Sample Preparation for Performing FAIRE on Adherent or Suspension Cells Without Large Fat Deposits

  Materials
  • Cultured cells: 1 × 106 – 5 × 107 cells for each experiment, preferably 1 × 107 cells
  • 37% (w/v) formaldehyde (Fisher Scientific, cat. no. F79‐500)
  • 2.5 M glycine (Fisher Scientific, cat. no. BP381‐500)
  • 1× PBS
  • 1× Dulbecco's phosphate buffered saline (Cellgro, cat. no. 21‐031)
  • Liquid nitrogen
  • Lysis buffer A (see recipe)
  • 10 µg/µl DNase‐free RNaseA (Roche, cat. no. 11119915001)
  • 20 mg/ml proteinase K (Roche, cat. no. 03115836001)
  • 10 mM Tris⋅Cl, pH 7.4 (Fisher Scientific, cat. no. BP152‐500)
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol (Sigma, cat. no. P3803)
  • 24:1 (v/v) chloroform/isoamyl alcohol (Sigma, cat. no. C0549)
  • 3 M sodium acetate, pH 5.2 (Mallinkrodt, cat. no. 7364)
  • 95% ethanol (Decon, cat. no. 2801)
  • 20 mg/ml glycogen (Roche, cat. no. 901393)
  • 70% ethanol (diluted from 95% ethanol), ice cold
  • Zymo DNA binding buffer (Zymo Research, cat. no. D4004‐1‐L)
  • Zymo wash buffer (Zymo Research, cat. no. D4003‐2‐4)
  • Nutator (BD/Clay Adams, cat. no. 421105)
  • Cell scrapers (Corning, cat. no. 3008)
  • 15‐ and 50‐ml conical tubes
  • Swinging bucket centrifuge with variable temperature (Fisher Scientific Accuspin 1R)
  • 2‐ml tubes with metal beads, 2.38 mm (MoBio, cat. no. 13117‐50)
  • Bead‐beater (Biospec Mini‐BeadBeater‐8)
  • Sonicator (Branson Sonifier 450D equipped with microtip)
  • 1.5‐ml microcentrifuge tubes
  • 37°, 55°, and 65°C incubators
  • SpeedVac, optional
  • Fluorometer with DNA quantification reagents and standards (Invitrogen, cat. no. Q32866) or NanoDrop ND‐1000
  • Zymo‐spin I columns (Zymo Research, cat. no. C1003‐250)
  • Additional reagents and equipment for agarose gel and ethidium bromide *A*(unit 2.5)
CAUTION: Formaldehyde is toxic by inhalation or if swallowed; is irritating to the skin, eyes, and respiratory system; and may be carcinogenic. Formaldehyde should be used with appropriate safety measures such as protective gloves, glasses, clothing, and sufficient ventilation. All waste should be handled according to hazardous waste regulations.CAUTION: Phenol/chloroform is harmful if swallowed or in contact with skin, causes severe skin burns and eye damage, is fatal if inhaled, and is potentially carcinogenic. It should be used with appropriate safety measures such as protective gloves, glasses, clothing, and sufficient ventilation. All waste should be handled according to hazardous waste regulations.CAUTION: Chloroform/isoamyl alcohol is harmful if swallowed, causes skin and eye irritation, and is potentially carcinogenic. It should be used with appropriate safety measures such as protective gloves, glasses, clothing, and sufficient ventilation. All waste should be handled according to hazardous waste regulations.

Support Protocol 1: Sample Preparation for Performing FAIRE on Adherent Lipid‐Laden Cells (or if Bead‐Beater is Unavailable)

  • Lysis buffer B (see recipe)
  • Sucrose pad (see recipe)
  • Tissue pulverizer (Biospec, cat. no. 59012N), mortar and pestle pre‐chilled in liquid nitrogen
  • Dounce (Kimble‐Chase, cat. no. 885300‐0000)
  • Serological pipets
CAUTION: Formaldehyde is toxic by inhalation or if swallowed; is irritating to the skin, eyes, and respiratory system; and may be carcinogenic. Formaldehyde should be used with appropriate safety measures such as protective gloves, glasses, clothing, and sufficient ventilation. All waste should be handled according to hazardous waste regulations.

Support Protocol 2: Sample Preparation for Performing FAIRE on Frozen Tissues

  • 20 to 200 mg frozen tissue, kept on dry ice to avoid thawing
  • Tissue pulverizer (Biospec, cat. no. 59012N), mortar and pestle pre‐chilled in liquid nitrogen
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Figures

Videos

Literature Cited

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   Patel, M., Simon, J.M., Iglesia, M.D., Wu, S.B., McFadden, A.W., Lieb, J.D., and Davis, I.J. 2012. Tumor‐specific retargeting of an oncogenic transcription factor chimera results in dysregulation of chromatin and transcription. Genome Res. 22:259‐270.
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   Simon, J.M., Giresi, P.G., Davis, I.J., and Lieb, J.D. 2012. Using formaldehyde‐assisted isolation of regulatory elements (FAIRE) to isolate active regulatory DNA. Nat. Protoc. 7:256‐267.
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Key References
  Giresi et al., 2007. See above.
  Describes the first application of FAIRE to human cells.
  Hogan et al., 2006. See above.
  The first comprehensive characterization of FAIRE as a method for regulatory elements isolation.
  Nagy et al., 2003. See above.
  Represents the first description of FAIRE in the literature (although officially not called FAIRE).
  Simon et al., 2012. See above.
  Presents more details about methods of detecting FAIRE enrichment, and first outlined the modifications for performing FAIRE on tissues or cells with significant lipid deposits.
  Song et al., 2011. See above.
  Compares FAIRE to other methods for isolation regulatory elements, including DNaset‐Hypersensitivity and Chlp.
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