Data were normalized using default settings in protocol GE2_107_Sep09 of Agilent's Feature Extractor software (version 10.7.1.1).
http://www.chem.agilent.com/Library/usermanuals/Public/G4813-90010_TwoColor_Prokaryote_Protocol_1.3.pdf (Parameters: Chamber type = OTHER: Agilent hybridization chambers, Quantity of label target used = 300, Mass unit = Nano gram, time = 17, Tiny time unit = hours, Volume = 40, Volume unit = Micro litre, temperature = 65)
Agilent Two-Color Microarray-Based Gene Expression (Quick Amp Labeling)Version: 5.7 Agilent publication number: G4140-90050 URL: http://www.chem.agilent.com/Library/usermanuals/Public/G4140-90050_Two-Color_GE_5.7.pdf. This document describes Agilent's recommended procedures for the preparation and labeling of complex biological targets and hybridization, washing, scanning, and feature extraction of Agilent 60-mer oligonucleotide microarrays for microarray-based two-color gene expression analysis. (Parameters: Scanning hardware = OTHER: Agilent Technologies Scanner G2505B US22502519, Scanning software = Feature Extraction Software [Agilent])
Aliquots containing approximately 10^7 CFU from a transposon mutant pool stored at -80C were diluted 1 to 1000 in 20 ml of BHI broth or BHI broth with 0.02% bile salts. Cells were grown at 37C for 20 hours, after which 1 ml of the bacteria cultures were spun down and used for the extraction of genomic DNA. (Parameters: start time = 20, time unit = hours, min temperature = 37, temperature unit = C, media = BHI)
Genomic DNA from mutant libraries was isolated using the Wizard Genomic DNA Purification kit (Promega), digested with AluI (New England Biolabs) and then purified on a Qiagen QIAquick PCR Purification column (Qiagen). 200 ng of the digested DNA was self-ligated by the Quick Ligation Kit (New England Biolabs) in a reaction volume of 20 ul. This ligation reaction was directly used as template for PCR amplification of the transposon–chromosome junctions with primer pair IPCR_AluI_F and IPCR_AluI_R in a reaction volume of 200 ul, using AccuPrime DNA polymerases (Invitrogen) with the following conditions: 94C for 1 min; 26 cycles of 94C for 18 sec, 56.5C for 30 sec, 68C for 50 sec; and 68C for 7 min. PCR products were purified using Qiagen QIAquick PCR Purification Kit. After purification, 200-500 ng DNA was redigested by AluI and used for in vitro transcription (IVT) in a volume of 20 ul using the T7 MEGAshortscript kit (Ambion) at 37°C for 6 hours. The RNA was first treated with DNase (Ambion) and then purified with the MEGAclear Kit (Ambion). (Parameters: Extracted product = genomic_DNA, Amplification = PCR)
5-10 ug of the purified RNA was used for generating labeled cDNA using the FairPlay III Microarray Labeling Kit (Agilent Technologies) as described in the manufacturers protocol. (Parameters: Amount of nucleic acid labeled = 5, Amplification = none, Mass unit = Micro gram)