strain: M4455 pull: 5 cbh2_copies: 0 growth rate: High
Biomaterial provider
ATCC
Treatment protocol
Three 2L chemostat reactors with a base media of Yeast Nitrogen Base supplemented with synthetic complete amino acids (Sigma) were inoculated with either the 2-copy (M3201), 6- copy (M4294) or control strain (M4455). The reactor was controlled at 35 °C with continuous flushing of nitrogen/carbon dioxide (95:5). After 3 exchanges of base media, a consistent growth rate of either 0.03 h-1 (low rate) or 0.3 h-1 (high rate) was achieved and a sample was taken for further analysis. After sampling, 3 exchanges of base media were allowed for the strains to re-establish a steady state. The chemostats were sampled again and this procedure was repeated for a total of 4 samples for the low growth reactor or 3 samples for the high growth reactor.
Growth protocol
Yeast Peptone Dextrose medium were used for general yeast growth, which was at a temperature between 30 °C and 35 °C. To establish a consistent growth rate, chemostats were utilized.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted using a previously described method that involved resuspending previously frozen cells in TRIzol reagent (Invitrogen, CA) and lysis via a bead beating method [23]. Briefly, frozen cell pellets were resuspended into 3 mL TRIzol reagent. Cells were lysed by adding ~1.5 mL of cell pellet/TRIzol mixture to 2 mL screw top tubes containing 0.25 g glass beads (MoBio Laboratories, CA) and bead beating at three 20 second cycles at 6500 rpm for several aliquots. Bead beating was conducted in a Precellys-24 (Bertin Technologies, Montigny-le-Bretonneux, France) bead beater. RNA was treated with DNAse I (Ambion, TX), purified using an RNeasy mini kit (Qiagen, CA), and quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, DE). The quality of extracted RNA was determined with an Agilent BioAnalyzer. Purified RNA of high quality was used as the template to generate ds-cDNA using Invitrogen Superscript double-stranded synthesis kit according to the manufacturers protocols (Invitrogen, CA). ds-cDNA was labeled, hybridized and washed according to the NimbleGen protocols essentially as described previously ( Yang S, Giannone RJ, Dice L, Yang ZK, Engle NL, Tschaplinski TJ, Hettich RL, Brown SD: Clostridium thermocellum ATCC27405 transcriptomic, metabolomic and proteomic profiles after ethanol stress. BMC Genomics 2012, 13:336.)
Label
Cy3
Label protocol
The labeling, hybridization, and scanning following NimbleGen company's protocols.
Hybridization protocol
The labeling, hybridization, and scanning following NimbleGen company's protocols.
Scan protocol
The labeling, hybridization, and scanning following NimbleGen company's protocols.
Description
R M4455 N=1 High chip ID: 451215 _22_Cy3 PM
Data processing
Statistical analysis was done with JMP Genomics 6.0 software (SAS Institute, Cary, NC). The data were subsequently normalized using the Loess normalization algorithm within JMP Genomics. An analysis of variance (ANOVA) was performed to determine differential expression levels between conditions and time points using the FDR testing method (p < 0.05).
