Cheryl Christianson, Dunham Lab, Princeton University
Extracted molecule
total RNA
Extraction protocol
Description : Filters were incubated for 1 hour at 65oC in lysis buffer (10 mM EDTA, 0.5% SDS, and 10 mM Tris, pH 7.5) and acid phenol. The aqueous phase was further extracted twice with an equal volume of chloroform using phase lock gel (Eppendorf). Total RNA was then ethanol precipitated and further purified over RNeasy columns (Qiagen). RNA quality was checked using the Bioanalyzer RNA Nano kit.
Label
Cy3
Label protocol
Description : 325 ng was used for microarray labeling with the Agilent Low RNA Input Fluorescent Linear Amplification Kit. Reactions were performed as directed except using half the recommended reaction volume and one quarter the recommended Cy-CTP amount. Dye incorporation and yield were measured with a Nanodrop spectrophotometer.
Cheryl Christianson, Dunham Lab, Princeton University
Growth protocol
Description : ATR Sixfors fermenters were modified for use as chemostats. Chemostat cultures were run at 30oC at a working volume of 300 mls, mixed at 400 rpm, and sparged at 5 standard liters per minute with humidified and filter-sterilized air. The dilution rate was set to 0.17 volumes/hour. Cultures were run in phosphate limited minimal defined medium containing the following (per liter): 100 mg calcium chloride, 100 mg sodium chloride, 500 mg magnesium sulfate, 5 g ammonium sulfate, 1 g potassium chloride, 500 mg boric acid, 40 mg copper sulfate, 100 mg potassium iodide, 200 mg ferric chloride, 400 mg manganese sulfate, 200 mg sodium molybdate, 400 mg zinc sulfate, 1 mg biotin, 200 mg calcium pantothenate, 1 mg folic acid, 1 mg inositol, 200 mg niacin, 100 mg p-aminobenzoic acid, 200 mg pyridoxine, 100 mg riboflavin, 200 mg thiamine, 50 mg adenine, 50 mg tryptophan, 20 mg uracil, 100 mg lysine, 20 mg methionine, 100 mg leucine, 100 mg G418, and 5 g glucose. Chemostats were inoculated with 1 ml overnight culture grown in chemostat media. Cultures were maintained in batch for 24 hours, at which time the media flow was switched on. Cultures were sampled daily for cell density by Coulter count, klett, and absorbance, and were considered to be in steady state when all parameters measurements were the same for two consecutive measurements, which occurred 4 days after inoculation for all cultures. 100 ml of cultures were harvested by vacuum filtration, flash-frozen in liquid nitrogen, and stored at ?80oC until RNA extraction.
Extracted molecule
total RNA
Extraction protocol
Description : Filters were incubated for 1 hour at 65oC in lysis buffer (10 mM EDTA, 0.5% SDS, and 10 mM Tris, pH 7.5) and acid phenol. The aqueous phase was further extracted twice with an equal volume of chloroform using phase lock gel (Eppendorf). Total RNA was then ethanol precipitated and further purified over RNeasy columns (Qiagen). RNA quality was checked using the Bioanalyzer RNA Nano kit.
Label
Cy5
Label protocol
Description : 325 ng was used for microarray labeling with the Agilent Low RNA Input Fluorescent Linear Amplification Kit. Reactions were performed as directed except using half the recommended reaction volume and one quarter the recommended Cy-CTP amount. Dye incorporation and yield were measured with a Nanodrop spectrophotometer.
Hybridization protocol
Description : Equal masses of differentially labeled control and sample cRNA were combined such that each sample contained at least 2.5 pmol dye. Samples were mixed with control targets, fragmented, combined with hybridization buffer, and applied to a microarray consisting of 60mer probes for each yeast open reading frame (Agilent). Microarrays were rotated at 60?C for 17 hours in a hybridization oven (Agilent). Arrays were then washed according to the Agilent SSPE wash protocol, and scanned on an Agilent scanner.
Scan protocol
Pixel Size : 10 Scan Date : 2006-04-18 Scan Time : 10:06:27 Scanner Make : Agilent Technologies Scanner Scanner Model : G2505B Scanning software : ChipScan Scanning software version : A.6.3.1
Description
former name: AA 2596 (cdc15-2) chemostat RNA
Data processing
Extraction Software : Agilent Feature Extractor Extraction Software Version : A.7.5.1 Datafile type : Agilent result file
