Technology - Location Analysis

Genome-wide location analysis was performed as previously described (Ren et al., 2000), except that crosslinking time was reduced to 30 minutes and high-resolution oligonucleotide arrays were used for hybridizations. Bound proteins were formaldehyde-crosslinked to DNA in vivo, followed by cell lysis and sonication to shear DNA. Crosslinked material was immunoprecipitated with the appropriate antibody, followed by reversal of the crosslinks to separate DNA from protein (Aparicio, 1999; Orlando et al., 2000). Immunoprecipitated DNA and reference DNA were amplified and differentially fluorescently labeled by ligation-mediated PCR. These samples were hybridized to a microarray consisting of spotted PCR products representing nearly all of the S.cerevisiae genome.

In analyzing histone modification, we find that an alternative experimental design yielded improved results. Specifically, material immunoprecipitated with antibodies directed against modified histones were compared to material immunoprecipitated with antibodies directed against core histones. The results of both approaches are analyzed with respect to transcriptional activity here.

Microarray design

The Agilent DNA microarray used here has 44,290 features consisting of 60-mer oligonulceotide probes. The array covers 12 Mb of the yeast genome (85%), excluding highly repetitive regions, with an average probe density of 266 bp. Intergenic regions are represented by 14,256 probes and ORFs are represented by 27,185 probes. The remaining 2,849 control features included blank spots and probes derived from Arabidopsis thaliana. The probe list for this array may be found here.