9 protocols
AccessionType
array scanning and feature extraction protocol
Title: Affymetrix CEL analysis. Description:
hybridization protocol
Affymetrix Generic Hybridization
bioassay_data_transformation
Title: Affymetrix CHP Analysis (ExpressionStat). Description:
nucleic_acid_extraction
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 -80°C. RNA quality was assessed with electrophoresis using a RNA bioanalyser 2100 and Lab RNAChip (Agilent Technologies) before progression to microarray hybridization.
specified_biomaterial_action
No treatment (control sample).
labeling
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.
grow
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.
specified_biomaterial_action
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.
specified_biomaterial_action
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.