In the oxidative stress and DNA damage experiments, cells were treated with 0.3mM H2O2 or 0.1% MMS, 25 or 40 minutes prior to transcription inhibition. At time point 0, the temperature of the culture was raised to 37 degrees by adding an equal amount of medium prewarmed to 49 degrees. This step inactivated the temperature-sensitive RNA polymerase II and therefore stopped transcription. Following transcription inhibition, 10 samples were taken for each time course (0, 5 min, 10 min, 15 min, 20 min, 30 min, 40 min, 50 min, 60 min) and a technical replicate of time point zero. In the second reference decay profile, the three last time points (40 min, 50 min, and 60 min) turned out to be outliers, and thus were not used and not submitted.
Growth protocol
Yeast strain Y262 (carrying a temperature-sensitive mutation in RNA polymerase II) was grown in YPD to mid-log phase in 26 degrees.
Extracted molecule
polyA RNA
Extraction protocol
Total RNA was extracted using MasterPure™ (EPICENTER Biotechnologies).
Label
biotin
Label protocol
cDNA was prepared using poly(T) primers, labeled cRNA was prepared using the GeneChip system according to manufacturer instructions.
Hybridization protocol
GeneChip system according to manufacturer instructions.
Scan protocol
GeneChip system according to manufacturer instructions.
Description
Decay profile following DNA damage, t=20 min
Data processing
RMA algorithm followed by normalization using spiked-in RNA as explained in detail in the supplementary of the paper (Shalem et al.).
