The preinoculum and inoculum preparation were performed as described before (Ren, C. et al. 2010. Metab Eng 12:446-54). Clostridium acetobutylicum ATCC 824 and clostridium acetobutylicum ATCC 824 ccpA mutant were grown anaerobically in P2 medium at 37 ºC. The fermentations were carried out in BioFlo 110 bioreactors (New Brunswick Scientific, Edison, NJ) with 1.5 L working volume, in which pH was stabilized at ≥5.0 by adding 9% (w/v) aqueous ammonia and mixed sugars (40 g/L D-glucose and 20 g/L D-xylose) were used as carbon sources.
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)
