The plasticity of nuclear architecture is controlled by dynamic epigenetic mechanisms including non-coding RNAs, DNA methylation, nucleosome repositioning as well as histone composition and modification. Here we describe a Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) protocol which allows the determination of chromatin accessibility in a reproducible, inexpensive, and straightforward manner.
Total time
~4–6 hours (plus overnight incubation step)
Model organism
Mammalian cultured cells (e.g., HEK293, mouse fibroblasts)
Steps
1
Perform formaldehyde crosslinking and harvest cells
Treat mammalian cells with formaldehyde to covalently crosslink DNA–protein complexes, then harvest cells by centrifugation. This preserves chromatin structure for downstream analysis.
▶ 01:03
2
Lyse cells and fragment chromatin
Lyse crosslinked cells and fragment chromatin using sonication or enzymatic digestion to generate nucleosome-sized DNA fragments. This step exposes nucleosome-free (accessible) DNA regions.
▶ 02:18
3
De-crosslink control DNA sample
Reverse formaldehyde crosslinks in a control DNA fraction by heating, releasing all DNA regardless of accessibility. This serves as a reference for total chromatin content.
▶ 03:25
4
Purify DNA using phenol-chloroform extraction
Remove proteins and contaminants from fragmented DNA via phenol–chloroform extraction and ethanol precipitation. This yields clean DNA suitable for qPCR or sequencing.
▶ 04:04
5
Quantify chromatin accessibility by qPCR
Measure enrichment of accessible DNA at regulatory regions using qPCR. Compare signal between crosslinked (accessible fraction) and control samples to determine chromatin accessibility.
▶ 06:44