|
| Status |
Public on Jun 15, 2016 |
| Title |
DG-8052 vs. WT-8052 at 24h replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
collected the whole fermentation cells, 24h
|
| Organism |
Clostridium beijerinckii NCIMB 8052 |
| Characteristics |
genotype/variation: degenerated strain
|
| Growth protocol |
WT-8052 and DG-8052 cells were grown at P2 medium in anaerobic chamber at 35 °C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total cellular RNA was extracted from homogenized cells using RiboPureTM RNA Purification Kit, bacteria (Ambion®, Life Technologies, Inc., US) according to the manufacturer’s instructions.
|
| Label |
Cy5
|
| Label protocol |
The Crystal Core® cDNA amplified RNA labelling kit (CapitalBio, Beijing, China) were used as the protocol. One µg of total RNA were primed with 1 µl of 100 µM T7 Oligo (dT) DNA primer at 70°C for 10 min to get the 1st strand cDNA, and then transcribed into cRNA at 37°C for 4 h in the presence of T7 enzyme Mix. Two µg of cRNA were reversed transcribed into cDNA at 25°C for 10min followed by 37°C for 1.5 h in the presence of 1.5 µl CbcScript II RTase (CapitalBio) with 4µl Random Primer. For each 1 µg of cDNA,100 µM each dATP, dTTP, dGTP, with 25 µM dCTP Cy3-label (Cy5-label) were used for labeling by 4µl Random Primer and1.2 µl Klenow Fragment .
|
| |
|
| Channel 2 |
| Source name |
collected the whole fermentation cells, 24h
|
| Organism |
Clostridium beijerinckii NCIMB 8052 |
| Characteristics |
genotype/variation: wild type
|
| Growth protocol |
WT-8052 and DG-8052 cells were grown at P2 medium in anaerobic chamber at 35 °C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total cellular RNA was extracted from homogenized cells using RiboPureTM RNA Purification Kit, bacteria (Ambion®, Life Technologies, Inc., US) according to the manufacturer’s instructions.
|
| Label |
Cy3
|
| Label protocol |
The Crystal Core® cDNA amplified RNA labelling kit (CapitalBio, Beijing, China) were used as the protocol. One µg of total RNA were primed with 1 µl of 100 µM T7 Oligo (dT) DNA primer at 70°C for 10 min to get the 1st strand cDNA, and then transcribed into cRNA at 37°C for 4 h in the presence of T7 enzyme Mix. Two µg of cRNA were reversed transcribed into cDNA at 25°C for 10min followed by 37°C for 1.5 h in the presence of 1.5 µl CbcScript II RTase (CapitalBio) with 4µl Random Primer. For each 1 µg of cDNA,100 µM each dATP, dTTP, dGTP, with 25 µM dCTP Cy3-label (Cy5-label) were used for labeling by 4µl Random Primer and1.2 µl Klenow Fragment .
|
| |
|
| |
| Hybridization protocol |
The labelled DNA was dissolved into hybridization buffer (2×GEx Hyb buffer (HI-RPM), Agilent In Situ Hybridization Kit; 25% formamide) at 45°C overnight. After hybridization, the slides were washed in 0.2% SDS and 2× SSC at 42°C for 5min, and then in 0.2× SSC at room temperature for 5min. Finally the slides were dried for microarray scan.
|
| Scan protocol |
Scanned on an Agilent G2565CA scanner
|
| Description |
Biological replicate 2 of 3. degenerated strain vs wild type strain, 24h culture cells
|
| Data processing |
Quantile normalized,background subtracted data obtained from log2 of processed Red signal/processed Green signal. Agilent software was used.
|
| |
|
| Submission date |
Nov 26, 2014 |
| Last update date |
Jun 15, 2016 |
| Contact name |
Yan Zhang |
| Organization name |
The Ohio State University
|
| Department |
Animal Sciences
|
| Street address |
1680 Madison Avenue
|
| City |
Wooster |
| State/province |
OH |
| ZIP/Postal code |
44691 |
| Country |
USA |
| |
|
| Platform ID |
GPL19469 |
| Series (1) |
| GSE63671 |
Clostridium beijerinckii NCIMB8052 wild-type cells vs.Clostridium beijerinckii NCIMB8052 degenerated strain cells |
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