CWB2plusFruc
Gordonia rubripertincta
Protocols: A pre-culture of G. rubripertincta CWB2 was grown at 30 °C in minimal media with fructose and styrene as sole source of carbon, respectively. After 5 days of cultivation the culture was diluted 1/10 in fresh media and incubated for 24 h with the respective substrate in a set of four Erlenmeyer flasks. Prior harvesting the cells, 10 % of an ice-cold STOP-solution (10 % buffered phenol in ethanol) was added to the culture followed by centrifugation at 11,000 x g for 5 minutes at 4 °C. The supernatant was discarded and the pellet was stored until RNA isolation at -80 °C. A pre-culture of G. rubripertincta CWB2 was grown at 30 °C in minimal media with fructose and styrene as sole source of carbon, respectively. After 5 days of cultivation the culture was diluted 1/10 in fresh media and incubated for 24 h with the respective substrate in a set of four Erlenmeyer flasks. To break up the cells, 150 µl of a 5 mg ml-1 lysozyme solution were added to the pellet, mixed and incubated at room temperature for 5 minutes. 450 µl of buffer RLT (Qiagen) and 50 mg of (0.1 mm) glass beads were added to resuspend and break the cells by repeated vortexing at 4 °C. The suspension was applied to QIAshredder column for homogenization and to remove particles from the sample. Extraction of total RNA Extraction was done by applying the RNeasy Mini Kit including on-column DNA digestion (Qiagen). Isolated RNA was stored at -80 °C and quality was controlled on the 2100 Bioanalyzer using the RNA 6000 Nano Kit (Agilent). RNA quality and quantity was again checked by an Agilent 2100 Bioanalyzer run (Agilent Technologies, Böblingen, Germany) and Trinean Xpose sytem (Gentbrugge, Belgium) prior and after rRNA depletion by Ribo-Zero rRNA Removal Kit (Bacteria) (Illumina, San Diego, CA, USA). TruSeq Stranded mRNA Library Prep Kit from (Illumina, (San Diego, CA, USA) was used to prepare the cDNA libraries to analyze the whole transcriptome.
ENA first public
2017-09-08
ENA last update
2017-08-29
External Id
SAMEA104229121
INSDC center alias
Technologieplattform Genomik, Centrum fur Biotechnologie (CeBiTec), Universitat Bielefeld, Universitatsstr. 27, Bielefeld, Germany.
INSDC center name
Technologieplattform Genomik, Centrum fur Biotechnologie (CeBiTec), Universitat Bielefeld, Universitatsstr. 27, Bielefeld, Germany.
INSDC first public
2017-09-08T17:01:26Z
INSDC last update
2017-08-29T14:40:18Z
INSDC status
public
Submitter Id
E-MTAB-6012:CWB2plusFruc
broker name
ArrayExpress
growth condition
fructose as sole source of carbon
sample name
E-MTAB-6012:CWB2plusFruc
strain
CWB2