array scanning and feature extraction protocol
Title: Affymetrix CEL analysis. Description:
Affymetrix Generic Hybridization
Title: Affymetrix CHP Analysis (ExpressionStat). Description:
RNA was obtained using the RNAeasy MIDI RNA extraction kit (Qiagen) using lysis buffer containing 0.1% beta-mercaptoethanol. RNA was extracted from lysates and purified according to the manufacturer’s standard protocol and subsequently stored at -80Â°C. RNA quality was assessed with electrophoresis using a RNA bioanalyser 2100 and Lab RNAChip (Agilent Technologies) before progression to microarray hybridization.
No treatment (control sample).
Gene expression was assessed on Affymetrix E. coli Genome GeneChip arrays (Affymetrix, Inc., Santa Clara, Calif.). Briefly, total RNA was prepared from E. coli cells by phenol-chloroform extraction, and ~10 ¼g of total RNA was used to synthesize cDNA. After purification with a Qiagen cDNA purification kit (Qiagen, Inc.), the cDNA was fragmented with DNase I. The fragmented cDNA was end biotinylated by terminal transferase with biotinylated ddATP.
Grown in Davis's Mineral Salts medium (Gibco BRL Life Technologies Inc.) with 0.1% D-glucose at 25 C (100 r.p.m. shaking) until an OD (550 nm) of 0.6.
Cell suspension was ammended with 1.37 M NaCl to obtain an osmotic pressure of 2.7 Os kg-1 for 10 minutes. The biomass was centrifuged at 4000 rpm and the pellet treated with RNAProtect (Qiagen) following the manufacturer's protocol.
Cell suspension was ammended with 1.25 M sucrose to an osmotic pressure of 2.7 Os kg-1 for 10 minutes. The biomass was centrifuged at 4000 rpm and the pellet treated with RNAProtect (Qiagen) following the manufacturer's protocol.