strain: M6880_2 biological rep: 2 sample date: 2014-01-31 tech rep y n: N parent adapted: Parent promot sample type: cDNA derived from Total RNA biomaterial provider: ORNL sample: 31 columnname: _16 intensity: PM
Treatment protocol
Two chemostats were used to grow each strain (M6880 and M7237) using growth conditions described above (growth protocol) (i.e. four chemostats in total) and each chemostat was sampled on the following dates; 1/28/14, 1/29/24, 1/30/14, 1/31/14.
Growth protocol
S. cerevisiae yeast strains M6880 and M7237 were cultured in chemostats using Verdyn’s medium with 10g/L glucose and 2g/L xylose as carbon sources. Chemostat cultures were operated at pH 5.5, 35C
Extracted molecule
total RNA
Extraction protocol
Trizol with bead beating extraction followed by DNaseI treatment and Qiagen kit purification 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
Label with Cy3 The labeling, hybridization, and scanning following NimbleGen company's protocols.
Hybridization protocol
NimbleGen Hyb Station, 42oC 24hours The labeling, hybridization, and scanning following NimbleGen company's protocols.
Scan protocol
Agilent Scanner_Green_100% The labeling, hybridization, and scanning following NimbleGen company's protocols.
Description
16
Data processing
Statistical analysis was done with JMP Genomics 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).
