I'm reading over the ENCODE Nature papers, and one of the papers referred to is "Global mapping of protein-DNA interactions in vivo by digital" by Hesselberth et al.
Genomic footprinting is a massively parallel variant of DNAse I hypersensitivity screening, which I feel I have a good handle on, but I don't understand this paragraph from the beginning of the results (the troublesome part is in bold):
To visualize regulatory protein occupancy across the genome of Saccharomyces cerevisiae, we coupled DNase I digestion of yeast nuclei with massively parallel DNA sequencing to create a dense whole-genome map of DNA template accessibility at nucleotide-level. We analyzed a single well-studied environmental condition, yeast a cells treated with the pheromone α-factor, which synchronizes cells in the G1 phase of the cell cycle. We isolated yeast nuclei and treated them with a DNase I concentration sufficient to release short (<300 bp) DNA fragments while maintaining the bulk of the sample in high molecular weight species (Supplementary Fig.1). These small fragments derive from two DNase I “hits” in close proximity, and therefore their isolation minimizes contamination by single fragment ends derived from random shearing. Because each end of the released DNase I ‘double-hit’ fragments represents an in vivo DNase I cleavage site, the sequence and hence genomic location of these sites can be readily determined by sequencing (Supplementary methods).
There's a reference to another paper by Sabo et al. about genome-scale DNase assays using microarrays in that paragraph that I'm reading, but if someone understands the biology well, I'd really appreciate an answer.
- Hesselberth JR, Chen X, Zhang Z, Sabo PJ, Sandstrom R, Reynolds AP, Thurman RE, Neph S, Kuehn MS, Noble WS, Fields S, Stamatoyannopoulos JA. 2009. Global mapping of protein-DNA interactions in vivo by digital genomic footprinting. Nature Methods 6(4): 283–289, doi:10.1038/nmeth.1313.
- Sabo PJ, Kuehn MS, Thurman R, Johnson BE, Johnson EM, Cao H, Yu M, Rosenzweig E, Goldy J, Haydock A, Weaver M, Shafer A, Lee K, Neri F, Humbert R, Singer MA, Richmond TA, Dorschner MO, McArthur M, Hawrylycz M, Green RD, Navas PA, Noble WS, Stamatoyannopoulos JA. 2006. Genome-scale mapping of DNase I sensitivity in vivo using tiling DNA microarrays. Nature Methods 3: 511-518, doi:10.1038/nmeth890.