strain: E50A treatment: None substrate: Avicel PH-101 5g/L harvest time (hrs post treatment): 0
Treatment protocol
Duplicate fermentations using MTC medium were conducted for Clostridium thermocellum strains, ATCC 27405 wild-type; ethanol adapted strain E50A (adapted to grow in presence of up to 50 g/L ethanol on Avicel); and ethanol adapted strain E50C (adapted to grow in presence of up to 50 g/L ethanol on cellobiose). These strains have been described previously by Shao X., et al Appl Microbiol Biotechnol (2011) 92:641–652. In this study, strains ATCC 27405 and E50A were grown by temperature and pH controlled Avicel fermentations and strain E50C by temperature and pH controlled cellobiose fermentations. At mid-log phase wild-type strain ATCC 27405 was shocked with 10g/L ethanol. At mid-log phase strain E50A was shocked with either 10 or 40 g/L ethanol. At mid-log phase strain E50C was shocked with either 40g/L ethanol. Samples were harvested at 0.25, 1, 2, and 4 h for transcriptomics analysis.
Growth protocol
Ruminiclostridium thermocellum ATCC 27405 and adapted strains were grown at 55 deg C in the 4L volume in 5L Twin BIOSTAT B Plus Fermenter in modified MTC medium as described by Shao et al., 2011.
Extracted molecule
total RNA
Extraction protocol
Briefly, samples were centrifuged and total cellular RNA was extracted by the TRIzol reagent (Invitrogen, Carlsbad, CA) and bead beating. Each total RNA preparation was treated with RNase-free DNase I (Ambion, Austin, TX) to digest residual chromosomal DNA and subsequently purified with the Qiagen RNeasy Mini kit in accordance with the instructions from the manufacturer. Total cellular RNA was quantified at OD260 and OD280 with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE) and the RNA quality was checked with Agilent Bioanalyzer (Agilent, CA). The purified RNA with good quality from each sample was used as the template to generate ds-cDNA using Invitrogen ds-cDNA synthesis kit (Invitrogen, CA).
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
5-L Twin BIOSTAT B Plus 4L Batch pH 7.0 100 rpm 55 deg C
Data processing
Microarrays were scanned with a SureScan High-Resolution DNA Microarray Scanner (5 μm) (Agilent), and the images were quantified using NimbleScan software (Roche NimbleGen, Madison, WI, USA). Raw data was log2 transformed and Loess normalized in JMP Genomics 5 (SAS Institute Inc., Cary, NC). We extracted probe data corresponding to genes while excluding probes for intergenic regions in our downstream analyses.
