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

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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 *undefined(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

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

Figure 21.26.1 Representative gel image showing varying degrees of sonication. NIH3T3 cells were fixed and lysed as described. Chromatin was then sheared by sonication for 0, 2, 4, 6, 8, and 10 cycles using the parameters outlined in step 2. Following pelleting of cell debris, crosslinks were reversed, and purified DNA was run on a 1% agarose gel. A 100‐bp ladder (lane marked M) is included for reference. The target range for fragment sizes is shown; six cycles yields an ideal distribution of fragment lengths. A high‐molecular‐weight band is slightly visible and marked with an asterisk.

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Figure 21.26.2 Expected results from FAIRE‐seq experiments. (A ) Genomic locus residing on chromosome 19 as visualized with the UCSC Genome Browser (Fujita et al., ) shows consistent FAIRE enrichment at transcriptional start sites (TSS) across seven ENCODE cell lines (Song et al., ). Data are presented as number of aligned, in silico extended reads per base, on a scale of 0 to 50 reads. Pink coloring atop tall peaks of enrichment represent where signal exceeded this range. (B ) Heatmap of normalized GM12878 FAIRE signal ± 3 kb around TSS ranked by gene expression in GM12878 cells. Color was assigned on a log₂ scale of −6 (background) to −2 (enriched). (C ) Average GM12878 FAIRE signal ± 3 kb around TSS across all genes. Enrichment peaks at around −125 bp. (D ) Average GM12878 FAIRE signal ± 3 kb around GM12878 CTCF sites, representing a class of distal regulatory elements.

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Videos

Literature Cited

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

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited