Title: Affymetrix CEL analysis. Description:
nucleic acid hybridization to array protocol
P-AFFY-3 GeneChip hybridization
Samples for RNA isolation were collected by pipetting the culture directly into 15-ml Falcon tubes containing 7.5 ml of icy-water to quickly chill the cells. Cells were harvested by spinning for 3-4 min at 6000 rpm, frozen in liquid N2 and stored at - 80C. Total cellular RNA was extracted using the RNeasy kit (Qiagen), following the manufacturer’s instructions with the spheroplasting protocol. Purified RNA samples were quantified by Qubit fluorometer (Invitrogen, Inc.) and Nanodrop spectrophotometer (Thermo Scientific, Inc.) (Parameters: Extracted product = total_RNA, Amplification = none)
Yeast strain BY4741 was grown overnight at 30C in YPD rich media. The yeast culture was diluted to an OD600 of 0.1 using fresh YPD media and further grown until an OD600 of 0.2. (Parameters: start time = 8, time unit = hours, min temperature = 30, temperature unit = C, media = YPD)
alpha-factor mating pheromone (GenScript) was added to a final concentration of 10 uM to allow cell synchronization at G1 phase, 7.5 ml samples were collected for RNA extraction while 40 ml samples were taken for nucleosomal DNA preparation and for flow cytometry (FACS) - microscope quality control analysis
normalization data transformation protocol
CEL files were imported into R/Bioconductor framework using affy package and justRMA function with default parameters, including quantile normalization. Reported intensity values were used as a relative measure of the different gene expression levels.
nucleic acid labeling protocol
cDNA synthesis, cRNA synthesis and labelling,were performed as described in the Affymetrix manual. Affymetrix (2000) Affymetrix GeneChip Expression Analysis Technical Manual. (Affymetrix, Santa Clara, CA).