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 phosphorothioate modificaiton genes mutant strain
Data processing
Raw data were normalized by Quantile algorithm, Gene Spring Software 11.0 (Agilent technologies, Santa Clara, CA, US).
