growth time: 12h serovar: Cerro 87 genotype: wild type
Treatment protocol
The cells were collected from theLB medium by centrifugation at 12,000 rpm for 1 min, resuspended in 1 mL TRIZOL Reagent (Cat#15596-018，Life technologies, Carlsbad, CA, US), and broken in a precellys homogenizer (6,500 rpm, 1×30 s; Peqlab) with glass beads (150–212 μm, Sigma). Cell debris was removed by centrifugation at 12,000 rpm for 5 min, and the extracted supernatants were stored at -80°C.
Growth protocol
Strains were grown at 37 ˚C on liquid Luria-Bertani (LB) medium. To obtain cells from different growth phases, the overnight bacterial cultures were diluted into 5 ml of LB medium at a concentration of 5000 cells/ml and the cell growth was monitored by the optical density 600 nm (OD600). The cells grown for 8h, 12h, 16h were considered to be at their early-, late-exponential and stationary phase, respectively.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using TRIZOL Reagent (Cat#15596-018，Life technologies, Carlsbad, CA, US) following the manufacturer’s instructions and checked for a RIN number to inspect RNA integration by an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, US).Qualified total RNA was further purified by RNeasy mini kit (Cat#74106, QIAGEN, GmBH, Germany) and RNase-Free DNase Set (Cat#79254, QIAGEN, GmBH, Germany).
Label
Cy3
Label protocol
Total RNA was amplified and labeled by Low Input Quick Amp Labeling Kit, One-Color (Cat#5190-2305, Agilent technologies, Santa Clara, CA, US), followed the manufacturer’s instructions. Labeled cRNA were purified by RNeasy mini kit (Cat#74106, QIAGEN, GmBH, Germany).
Hybridization protocol
Each Slide was hybridized with 1.65μg Cy3-labeled cRNA using Gene Expression Hybridization Kit (Cat#5188-5242, Agilent technologies, Santa Clara, CA, US) in Hybridization Oven (Cat#G2545A, Agilent technologies, Santa Clara, CA, US), according to the manufacturer’s instructions. After 17 hours hybridization, slides were washed in staining dishes (Cat#121, Thermo Shandon, Waltham, MA, US) with Gene Expression Wash Buffer Kit(Cat#5188-5327, Agilent technologies, Santa Clara, CA, US), followed the manufacturer’s instructions.
Scan protocol
Slides were scanned by Agilent Microarray Scanner (Cat#G2565CA, Agilent technologies, Santa Clara, CA, US) with default settings，Dye channel: Green ,Scan resolution=5μm, PMT 100%，10% ，16bit. Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, US)
Description
Gene expression of wild-type strain
Data processing
Raw data were normalized by Quantile algorithm, Gene Spring Software 11.0 (Agilent technologies, Santa Clara, CA, US).
