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])
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)
For RNA isolation, 2 ml of each culture were transferred into an Eppendorf tube and spun down at 13,000 × g for 20 s and the cell pellets were snap-frozen in liquid nitrogen. The time between removal from the incubator and freezing of the cell pellets was approximately 60 s. Within 20 min after freezing, 1 ml of TRI reagent (Ambion) was added to the frozen pellets and the suspension was transferred into a 2-ml tube filled with 0.5g of 0.1 mm zirconia/silica beads (Biospec) . Cells were disrupted by beadbeating three times for 1 min with intermittent cooling on ice. RNA was then extracted following Ambion’s TRI reagent protocol. Residual chromosomal DNA was removed by treating samples with the TURBO DNA-free kit (Ambion). Extracted RNA samples were quantified using a Nanodrop 1000 spectrophotometer (Isogen Life Science) and stored in 70% ethanol-83 mM sodium acetate buffer (pH 5.2) at -80C.(Parameters: Extracted product = total_RNA, Amplification = none)
Strains were cultured overnight in 3 ml of BHI broth in 10 ml tubes at 37C with aeration by rotary shaking at 150 rpm and pre-warmed BHI broth (25 ml in 50-ml Falcon tube) was inoculated with the overnight culture to a starting absorbance at 660 nm (OD660) of 0.025 and incubated at 37C as described above. When OD660 reached 0.3 (mid-exponential growth phase), 1 ml of the culture was transfered into a eppendorf tube and freezed immediately in liquid nitrogen for RNA isolation. Meanwhile, bile salts was added (with final concetration 0.02%) into the rest of the culture. At time points of 5 minutes and 15 minutes after adding bile salts, 1 ml of the culture was transfered and used for isolation as described above. (Parameters: start time = 5, stop time = 15, time unit = minutes, min temperature = 37, temperature unit = C, media = BHI)