Cultures (500 mL) in 1L 45-mm-diameter screw top bottles were inoculated with cells in exponential phase to a density of 1 x 10^6 cells/ml. Cells were harvested when the culture reached mid-log phase, typically around 3-4 x 10^7 cells/ml. In the case of biomass cultures, harvested culture was first filtered through a coffee filter into chilled centrifuge bottles, then rapidly cooled in an ethanol-dry ice bath, after which the cells were centrifuged at 5,000 rpm for 15 minutes and the cell pellet was stored at -80°C.
Growth protocol
C. saccharolyticus (DSM 8903) was obtained as a freeze-dried culture from the German Collection of Microorganisms and Cell Cultures (DSMZ [http://www.dsmz.de]). After reanimation, C. saccharolyticus was maintained on DSMZ640 medium at 70˚C with the following changes: Trypticase, FeCl3 x 6H2O, resazurin and Cysteine-HCl x H2O were omitted and 0.5% (w/v) Na2S x 9H2O was added as a reducing agent. Polysaccharides and biomass used include: crystalline cellulose (Avicel PH-101, FMC); glucomannan (konjac); pectin (practical grade); birchwood xylan (Sigma); dilute acid pretreated switchgrass (Panicum virgatum, –20/+80 mesh fraction; pretreatment was in a Sunds reactor at the National Renewable Energy Laboratory [NREL]), dilute acid pretreated poplar (P. trichocarpa x deltoides, provided by NREL), P. trichocarpa and genetically modified as4CL, 7-1 and 7-2 P. trichocarpa (ground to 80 mesh). For the biomass microarray experiment, all polysaccharides and biomass were added at a concentration of 1 g/L, noting that acid-treated poplar and switchgrass were added at a wet weight. All cultures were sub-cultured four times on the applicable substrate in 50mL batch cultures under N2 headspace in 125 ml serum bottles prior to inoculation for cell harvesting. Cell densities (cells/ml) were monitored using epifluorescence microscopy.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted using TRIzol reagent (Invitrogen) and columns from a RNeasy kit (Qiagen). Biological repeats were used for all conditions, and total RNA was pooled prior to the reverse transcriptase reaction.
Label
cy3
Label protocol
10 ug RNA was mixed with 6 ug random primers, and primed at 70˚C for 10 min. Reverse transcription used Superscript III (Invitrogen) and a dNTP mixture: dATP, dCTP, dGTP, dTTP, aa-dUTP (1:1:1:0.33:0.67). Samples were incubate at 46 ˚C overnight. cy-dyes (GE Healthcare) were resuspend in DMSO and added to cDNA resuspended in 4.5 uL of 0.1M sodium carbonate buffer, pH 9.0. Labeling was allowed to proceed for 2 hr in th dark. cy-dye labeled cDNA was then cleaned using a Qiagen PCR clean up column.
Cultures (500 mL) in 1L 45-mm-diameter screw top bottles were inoculated with cells in exponential phase to a density of 1 x 10^6 cells/ml. Cells were harvested when the culture reached mid-log phase, typically around 3-4 x 10^7 cells/ml. In the case of biomass cultures, harvested culture was first filtered through a coffee filter into chilled centrifuge bottles, then rapidly cooled in an ethanol-dry ice bath, after which the cells were centrifuged at 5,000 rpm for 15 minutes and the cell pellet was stored at -80°C.
Growth protocol
C. saccharolyticus (DSM 8903) was obtained as a freeze-dried culture from the German Collection of Microorganisms and Cell Cultures (DSMZ [http://www.dsmz.de]). After reanimation, C. saccharolyticus was maintained on DSMZ640 medium at 70˚C with the following changes: Trypticase, FeCl3 x 6H2O, resazurin and Cysteine-HCl x H2O were omitted and 0.5% (w/v) Na2S x 9H2O was added as a reducing agent. Polysaccharides and biomass used include: crystalline cellulose (Avicel PH-101, FMC); glucomannan (konjac); pectin (practical grade); birchwood xylan (Sigma); dilute acid pretreated switchgrass (Panicum virgatum, –20/+80 mesh fraction; pretreatment was in a Sunds reactor at the National Renewable Energy Laboratory [NREL]), dilute acid pretreated poplar (P. trichocarpa x deltoides, provided by NREL), P. trichocarpa and genetically modified as4CL, 7-1 and 7-2 P. trichocarpa (ground to 80 mesh). For the biomass microarray experiment, all polysaccharides and biomass were added at a concentration of 1 g/L, noting that acid-treated poplar and switchgrass were added at a wet weight. All cultures were sub-cultured four times on the applicable substrate in 50mL batch cultures under N2 headspace in 125 ml serum bottles prior to inoculation for cell harvesting. Cell densities (cells/ml) were monitored using epifluorescence microscopy.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted using TRIzol reagent (Invitrogen) and columns from a RNeasy kit (Qiagen). Biological repeats were used for all conditions, and total RNA was pooled prior to the reverse transcriptase reaction.
Label
cy5
Label protocol
10 ug RNA was mixed with 6 ug random primers, and primed at 70˚C for 10 min. Reverse transcription used Superscript III (Invitrogen) and a dNTP mixture: dATP, dCTP, dGTP, dTTP, aa-dUTP (1:1:1:0.33:0.67). Samples were incubate at 46 ˚C overnight. cy-dyes (GE Healthcare) were resuspend in DMSO and added to cDNA resuspended in 4.5 uL of 0.1M sodium carbonate buffer, pH 9.0. Labeling was allowed to proceed for 2 hr in th dark. cy-dye labeled cDNA was then cleaned using a Qiagen PCR clean up column.
Hybridization protocol
Sample preparation steps for microarray hybridization: Prepare Prehyb buffer: 225mL water, 75mL 20X SSC, 0.3g SDS, 0.3g BSA. Preheat Prehyb buffer 45 min 42 degree C. Prehybidize slides at 42 degree C for 45 min. Wash slides by dipping 5 time in sterile dH2O at RT. Dip twice in isopropanol at RT. Dry slide in slide spinner. Add 1 ul COT1-DNA (20mg/ml) to samples. Heat probe mixture to 95oC in heat block for 2 min to denature DNA, do a quick spin. Add 30 ul of 2x hyb. buffer that has been preheated to 42oC to each tube. Hybridization buffer: 27.2uL water, 20uL 20X SSC, 8uL 50X Denhardts solution, 24uL formamide, 0.8uL 10% SDS. Apply hyb. mix to slide and cover with a 20x60 mm polyethylene hydrophobic coverslip. Place slide in water proof container, cover well with foil and place in 42 degree C bath for 18-20 hours. The following is a standard hybridization method that seems to work well for this procedure: 1 wash cycle at 42 degree C with 2X SSC w/ 0.2% SDS. 1 wash cycle at RT with 0.5X SSC w/ 0.2% SDS. 3 wash cycles at RT with 0.5X SSC I
Scan protocol
Images were aquired with a GenePix 4000B scanner (Molecular Devices) using 532 nm and 635 nm lasers simultaneously. An automatic photomultiplier (PMT) gain adjustment was used to balance the 532 nm and 635 nm channels prior to scanning the whole slide. A pixel size of 10 microns was selected for the image resolution. Separate images were saved for the 532 and 635 nm scans, and analyzed together using GenePix Pro (v6.0) software.
Description
Loop #1
Data processing
Signals for each channel were obtained using GenePix Pro (v6.0) using circular features and were background substrated using the median local intensity around the spot. Once all the slides were quantitated, data from the dye-flip was analyzed with JMP Genomics 5.0 (SAS, NC), normalized using ANOVA (Loop #3), Quantiles (Loop #1) or Lowess (Loop #2) then ANOVA to estimate the global fixed effects of dye & treatment and the random effects array and block followed by using a mixed effects ANOVA model [Wolfinger et al. J Comput Biol 8 625-637 (2001).
