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. 2012 Jan 19;7(2):256-67.
doi: 10.1038/nprot.2011.444.

Using formaldehyde-assisted isolation of regulatory elements (FAIRE) to isolate active regulatory DNA

Jeremy M Simon  1 , Paul G Giresi, Ian J Davis, Jason D Lieb
Affiliations

Using formaldehyde-assisted isolation of regulatory elements (FAIRE) to isolate active regulatory DNA

Jeremy M Simon et al. Nat Protoc. .

Erratum in

  • Nat Protoc. 2014 Feb;9(2):501-3

Abstract

Eviction or destabilization of nucleosomes from chromatin is a hallmark of functional regulatory elements in eukaryotic genomes. Historically identified by nuclease hypersensitivity, these regulatory elements are typically bound by transcription factors or other regulatory proteins. FAIRE (formaldehyde-assisted isolation of regulatory elements) is an alternative approach to identify these genomic regions and has proven successful in a multitude of eukaryotic cell and tissue types. Cells or dissociated tissues are cross-linked briefly with formaldehyde, lysed and sonicated. Sheared chromatin is subjected to phenol/chloroform extraction and the isolated DNA, typically encompassing 1-3% of the human genome, is purified. We provide guidelines for quantitative analysis by PCR, microarrays or next-generation sequencing. Regulatory elements enriched by FAIRE have high concordance with those identified by nuclease hypersensitivity or chromatin immunoprecipitation (ChIP), and the entire procedure can be completed in 3 d. FAIRE has low technical variability, which allows its usage in large-scale studies of chromatin from normal or diseased tissues.

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Figures

Figure 1
Figure 1
Example timeline for FAIRE protocol. Steps are grouped by day for the typical timeline, but utilizing Pause Points will extend the duration.
Figure 2
Figure 2
Representative gel image showing varying degrees of sonication. NIH3T3 cells were fixed and lysed as described above. Chromatin was then sheared by sonication for 0, 2, 4, 6, 8, and 10 cycles using the parameters outlined in step 2A. After clearing 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; fewer than six cycles of sonication is insufficient for solubilization and shearing of chromatin, whereas sonication beyond six cycles leads to oversonication. A high molecular weight band is slightly visible and marked with an asterisk.
Figure 3
Figure 3
Expected results from FAIRE-seq experiments. A. Genomic locus residing on chromosome 19 as visualized with the UCSC Genome Browser shows consistent FAIRE enrichment at transcriptional start sites (TSS) across seven ENCODE cell lines. 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 ±3kb 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 ±3kb around TSS across all genes. Enrichment peaks around −125bp. D. Average GM12878 FAIRE signal ±3kb around GM12878 CTCF sites, representing a class of distal regulatory elements.
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