5 protocols
hybridization protocol
Affymetrix Generic Hybridization
First-strand cDNA was purified using the MinElute PCR purification kit (Qiagen) and 5 ug were fragmented and labeled using the GeneChip WT Terminal labeling kit (Affymetrix) according to manufacturers protocol. The labelled cDNA samples were denatured in a volume of 300 ul containing 50 pM control oligonucleotide B2 (Affymetrix) and Hybridization mix (GeneChip Hybridization, Wash and Stain kit, Affymetrix) of which 250 ul were hybridized per array (S. cerevisiae yeast tiling array, Affymetrix, PN 520055). Hybridizations were carried out at 45C for 16 h with 60 rpm rotation. The staining was carried out using the GeneChip Hybridization. Wash and Stain kit with fluidics protocol FS450_0001 in an Affymetrix Fluidics station
Yeast strains were streaked for 3 days on YEPD plates at 25C. Cells were grown in 5ml SC complete preculture at 25C, diluted into 50ml SC complete and exponentially grown overnight to OD600=0.8.
Cells were harvested and total RNA was extracted following the hot phenol procedure. Cells were lysed by vortexing with glass beads in presence of hot acid phenol. RNA was then cleaned by phenol:chloroform extraction followed by ethanol precipitation. cDNA was sythesized in the presence of actinomycin D according to a detailed procedure described in Materials and Methods.
Actinomycin D- to ensure strand specificity of the tiling array data (to inhibit second strand synthesis during the amplification step).