w303 pump mutants (MATa can1-100, his3-11,15, leu2-3,112, trp1-1, ura3-1, ade2-1, pdr1::kanMX, pdr3::kanMX), containing two copies (pRS304GAL- a-syn -GFP, pRS306GAL- a-syn -GFP) of alpha synuclein fused to Green Fluorescent Protein.
Extracted molecule
total RNA
Extraction protocol
hot phenol-chloroform extraction (Schmitt et al., 1990)
Label
Cy3
Label protocol
MICROMAX ASAP RNA Labeling Kit (Perkin Elmer)
Hybridization protocol
Gene Expression Hybridization Kit (Agilent)
Scan protocol
GenePix 4000B
Description
Yeast cells were induced to a starting O.D. of 0.3 in galactose media and harvested at 0, 2, 4, and 6 hours post-induction (50 ml aliquot per timepoint). For each comparison, 3 pairs of biological replicates were hybridized to 3 different arrays. Labeling, hybridization and scanning performed by the Whitehead Institute Center for Microarray Technology.
Data processing
Data were analyzed using the limma package from Bioconductor. Spots were background corrected using a normal and exponential convolutional model and an offset of 50 was added. After loess normalization by print tip, log2 ratios were calculated. Differential expression was assayed at each timepoint using a moderated t-test, corrected for false discovery rate.
