HOMESEARCHCONTACT US
ResearchPersonnelPublications

Activated Signal Transduction Kinases Frequently Occupy Target Genes

Study Design

Data Insights Supplemental Material

Acknowledgements

Technology - Location Analysis
Outline of Procedure

Genome-wide location analysis was performed as previously described (Ren et al., 2000), except that crosslinking time was reduced to 90 minutes at room temperature followed by 90 minutes at 4°C. and high-resolution oligonucleotide arrays were used for hybridizations. Bound proteins were crosslinked to DNA in vivo, followed by cell lysis and shearing of DNA by sonication. 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 high-resolution oligonucleotide arrays (see below).

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.

 
COLLABORATORSINTERNAL SITEQUICK LINKS
   
YOUNG LAB
Whitehead Institute
9 Cambridge Center
Cambridge, MA 02142
[T] 617.258.5218
[F] 617.258.0376
CONTACT US