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. -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 = Genome-wide binding of Fhl1 and Ifh1
