growth condition: cultured in chemically defined medium (CDM) supplemented with serum(S) and epinephrine (Epi) to mid-log phase
Treatment protocol
For serum treated A. pleuropneumoniae samples, filtered cattle serum at the concentration of 1/40 was added into CDM, for catecholamines treated A. pleuropneumoniae samples, epinephrine (Epi), norepinephrine (NE) and dopamine (DA) at 50μM were added respectively into CDM containing 1/40 serum.
Growth protocol
A. pleuropneumoniae 4074 were cultured in chemically defined medium (CDM) at 37°C with rotation at 200 rpm.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using RNA-Solv Reagent (Omega) according to the manufacturer’s instructions, then purified using QIAGEN RNeasy Mini Kit (QIAGEN).
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), following the manufacturer’s instructions. Labeled cRNA were purified by RNeasy mini kit (Cat#74106, QIAGEN, GmBH, Germany).
Hybridization protocol
Each Slide was hybridized with 600ng 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.
Description
biological replicate 2 of 3 for A. pleuropneumoniae cultured in CDM plus serum and Epi
Data processing
Data were extracted with Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, US). Raw data were normalized by Quantile algorithm, Gene Spring Software 11.0 (Agilent technologies, Santa Clara, CA, US).
