BioProject

Array design -Platform: amino-silane coated glass slides (GAPS II, Corning) -S. More...

Array design -Platform: amino-silane coated glass slides (GAPS II, Corning) -S. cerevisiae intergenic regions amplified from S288C genomic DNA (ResGen) using the intergenic region primer oligonucleotides (ResGen) (Harismendy et al. EMBO J. 22(18): 4738-4747, 2003). The primers allow the amplification of the sequence located on either side of elements such as open reading frames, tRNAs, small nuclear RNAs, Ty elements, solo d, etc. A complete description of the primers can be obtained on the web site (ftp://ftp.resgen.com/pub/genepairs/yeast_intergenic). -The PCR products were purified and spotted using an automated arrayer (MicroGrid II, BioRobotics) Experiment design -Type of experiment: genome wide binding analysis -Experimental factor: genotype (Wild-Type, Fhl1-myc, Ifh1-myc); drug (rapamycin) -Number of hybridization performed in the experiment: 15 (four conditions with three to five biological replicates each) -Reference used for each condition: ChIP from the wild-type strain -Quality control: one dye-swap per condition Samples used, extract preparation and labeling -The origin of the biological sample: o Species: S. cerevisiae o Cell type: (1) W303; (2) W303+IFH1-13myc-TRP1MX6;(3) W303+FHL1-13myc-HIS3MX6. -Growth conditions: log phase (2x107/mL) at 30ºC in YPD medium; when indicated rapamycin was added at the final concentration of 200 ng/mL for 90 min. -Cross-linking for 30 min at 30ºC -Chromatin immunoprecipitation: Dynabeads M280 coupled to sheep anti-mouse IgG or sheep anti-rabbit IgG and mouse monoclonal antibodies against the myc epitope (culture supernatant of 9E10) were used for immunoprecipitation according to standard techniques. -Labeling protocol(s): each IP was blunt-ended, ligated to linker DNA, then subjected to Ligation Mediated (LM)-PCR in the presence of amino-allyl dUTP. Labeling was performed for 1 hour at room temperature in the presence of NHS-ester Cy3 or Cy5 in 0.1 M Sodium Carbonate. The labeling reaction was stopped with 2 M Hydroxylamine and the labeled DNA purified. Hybridization procedures and parameters -Standard protocols were used Measurement data and specifications -Software for scanning: GenePix Pro 4.0 (Axon Instruments, Inc.) -Scanner: GenePix 4000A (Axon Instruments, Inc.) Keywords: dose response Less...