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.
Due to their biochemical properties, the DNA fragments cross-linked to nucleosomes will preferentially sit in the organic phase.
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.
[2][3] In addition, a reference or input genome, which has not been cross-linked, is often sequenced alongside to determine the level of background noise.
BedTools[11] is used to merge the enriched regions residing close to each other to form COREs (Cluster of open regulatory elements).
This helps in the identification of chromatin accessible regions and gene regulation patterns which would have been undetectable otherwise, considering the lower resolution FAIRE-seq often brings with it.
[12] The major limitation of this method, i.e. the low signal-to-noise ratio compared to other chromatin accessibility assays, makes the computational interpretation of these data very difficult.
[14][15] The subsequently developed ATAC-seq employs the Tn5 transposase, which inserts specified fragments or transposons into accessible regions of the genome to identify and sequence open chromatin.