FAIRE-Seq

FAIRE-Seq (Formaldehyde-Assisted Isolation of Regulatory Elements) is a method in molecular biology used for determining the sequences of those DNA regions in the genome associated with regulatory activity. The technique was developed in the laboratory of Jason D. Lieb at the University of North Carolina, Chapel Hill. In contrast to DNase-Seq, the FAIRE-Seq protocol doesn't require the permeabilization of cells or isolation of nuclei, and can analyse any cell types. In a study of seven diverse human cell types, DNase-seq and FAIRE-seq produced strong cross-validation, with each cell type having 1-2% of the human genome as open chromatin.

The protocol is based on the fact that the formaldehyde cross-linking is more efficient in nucleosome-bound DNA than it is in nucleosome-depleted regions of the genome. This method then segregates the non cross-linked DNA that is usually found in open chromatin, which is then sequenced. The protocol consists of cross linking, phenol extraction and sequencing the DNA in aqueous phase.

FAIRE uses the biochemical properties of protein-bound DNA to separate nucleosome-depleted regions in the genome. Cells will be subjected to cross-linking, ensuring that the interaction between the nucleosomes and DNA are fixed. After sonication, the fragmented and fixed DNA is separated using a phenol-chloroform extraction. This method creates two phases, an organic and an aqueous phase. Due to their biochemical properties, the DNA fragments cross-linked to nucleosomes will preferentially sit in the organic phase. Nucleosome depleted or ‘open’ regions on the other hand will be found in the aqueous phase. By specifically extracting the aqueous phase, only nucleosome-depleted regions will be purified and enriched.

FAIRE-extracted DNA fragments can be analyzed in a high-throughput way using next-generation sequencing techniques. In general, libraries are made by ligating specific adapters to the DNA fragments that allow them to cluster on a platform and be amplified resulting in the DNA sequences being read/determined, and this in parallel for millions of the DNA fragments.

Depending on the size of the genome FAIRE-seq is performed on, a minimum of reads is required to create an appropriate coverage of the data, ensuring a proper signal can be determined. In addition, a reference or input genome, which has not been cross-linked, is often sequenced alongside to determine the level of background noise.

...
Wikipedia