genotype: E. coli DH1 containing 3 plasmids pMevT(C159A), pMevB, pC9b treatment: control
Treatment protocol
When the OD reached 0.3, 0.5 mM IPTG was added to all flasks to induce the pathway protein expression. Also at this point, 0.2% v/v isopentenol was added to the 3 test flasks. 2.5 hr later the samples were extracted.
Growth protocol
E. coli DH1 containing 3 plasmids: pMevT(C159A), pMevB, pC9b, was inoculated to an OD of 0.1 into 250 ml flasks containing 60 ml M9 media and grown at 30 C.
Extracted molecule
total RNA
Extraction protocol
Samples were snap frozen in liquid nitrogen and the cells were lysed with 15 mg/ml lysozyme and total RNA was extracted with the Rneasy Mini Kit. DNA was removed with an on-column DNase treatment. RNA integrity was verified with a 2100 Bioanalyzer (Agilent)
Label
Alexa Fluor 555
Label protocol
20 ug of total RNA was converted to cDNA and labeled with Alexa Fluor 555 with the Superscript Plus Indirect cDNA labeling kit (Invitrogen)
Hybridization protocol
Hybridization was performed by NimbleGen Systems Inc., Madison, WI, USA following their standard operating protocol. See www.nimblegen.com.
Scan protocol
Scanning was performed by NimbleGen Systems Inc., Madison, WI USA, following their standard operating protocol. See www.nimblegen.com.
Description
Biological replicate 3 of 3
Data processing
The raw data (.pair file) was subjected to RMA (Robust Multi-Array Analysis; Irizarry et al. Biostatistics 4(2):249), quantile normalization (Bolstad et al. Bioinformatics 19(2):185), and background correction as implemented in the NimbleScan software package, version 2.4.27 (Roche NimbleGen, Inc.).
