10 days-old light-grown Arabidopsis ein5-1 seedlings were transferred to a Petri-plate containing incubation buffer (1mM Pipes, 1mM sodium citrate, 1mM KCl, 15mM sucrose), and maintained in agitation at 75 rpm for 30 min covered with foil. At this point vacuum was applied for exactly 15 seconds and cordycepin (Sigma Chemical Co., St. Louis) was added to a final concentration of 150 ug/ml. The seedlings were then incubated for 2 more hours in the dark at room temperature and kept in agitation at 75 rpm in Petri-plates. The seedlings were then briefly blotted to on filter paper to dry and kept in liquid nitrogen.
Growth protocol
10 days-old light-grown
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from the samples using the RNAeasy plant kit (Qiagen, Valencia, CA).
Label
Biotinylated
Label protocol
Total RNA was extracted from the samples using the RNAeasy plant kit (Qiagen, Valencia, CA). Biotinylated target RNA was prepared from 120 ug of total RNA from each sample using the GeneChip® Expression Analysis system (Affymetrix, Santa Clara, CA).
Hybridization protocol
Each sample of the purified and fragmented labeled target cRNA was hybridized to one set of Arabidopsis genome tiling arraysconsisting of twelve custom made arrays (Yamada, K et al., 2003). Hybridization, washes and the staining were carried out as described above for the Affymetrix array ATH1.
Scan protocol
GeneChips were scanned on Affymetrix scanner G7
Description
For the oligonucleotide tiling array experiments 10 days-old light-grown Arabidopsis ein5-1 seedlings were transferred to a Petri-plate containing incubation buffer (1mM Pipes, 1mM sodium citrate, 1mM KCl, 15mM sucrose), and maintained in agitation at 75 rpm for 30 min covered with foil. At this point vacuum was applied for exactly 15 seconds and cordycepin (Sigma Chemical Co., St. Louis) was added to a final concentration of 150 ug/ml. The seedlings were then incubated for 2 more hours in the dark at room temperature and kept in agitation at 75 rpm in Petri-plates. The seedlings were then briefly blotted to on filter paper to dry and kept in liquid nitrogen. Total RNA was extracted from the samples using the RNAeasy plant kit (Qiagen, Valencia, CA). Biotinylated target RNA was prepared from 120 ug of total RNA from each sample using the GeneChip® Expression Analysis system (Affymetrix, Santa Clara, CA). Each sample of the purified and fragmented labeled target cRNA was hybridized to one set of Arabidopsis genome tiling arrays consisting of twelve custom made arrays (Yamada, K et al., 2003). Hybridization, washes and the staining were carried out as described above for the Affymetrix array ATH1. Cell files were obtained using the GCOS software (Affymetrix, Santa Clara, CA) and the ChipViewer software was used to visualize and analyze the tiling chip data (Yamada et al. 2003. Empirical Analysis of Transcriptional Activity in the Arabidopsis Genome. Science 302, 842-846). Data submitted by Huaming Chen. Contributors: Gabriela Olmedo, Hongwei Guo, Brian D. Gregory, Saeid D. Nourizadeh, Laura Aguilar-Henonin, Hongjiang Li, Fengying An, Plinio Guzman, and Joseph R. Ecker
Data processing
CEL files were obtained using the GCOS software (Affymetrix, Santa Clara, CA) and the ChipViewer software was used to visualize and analyze the tiling chip data (Yamada et al. 2003. Empirical Analysis of Transcriptional Activity in the Arabidopsis Genome. Science 302, 842-846).
