|
| Status |
Public on Jul 29, 2010 |
| Title |
pMG1_IrrE 10 min, biological rep1 |
| Sample type |
RNA |
| |
|
| Source name |
E. coli K12 strain JM109 harboring pMG1-IrrE
|
| Organism |
Escherichia coli |
| Characteristics |
cell line: JM109
|
| Treatment protocol |
Treatment protocol- E.coli cells with pMG1 grown in LB media with at 37º C until early log phase (0.4), then were treated with 1 mol/L NaCl for 0min,10 min,60min,after that the cells were harvested to extract RNA.
|
| Growth protocol |
E.coli cells with pMG1 grown with aeration in LB media at 37º C until early log phase (0.4) and were treated with 1 mol/L NaCl for 10 min, after that the cells were harvested to extract RNA. LB medium recipe is as follow: 10g tryptone, 5g yeast extract, 10g NaCl per 1 liter.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the commercial product TRIzol Reagent (Invitrogen, Carlsbad, CA).Disrupt about 107cells with a homogenizer with 1ml TRIzol Reafent on ice. Store the homogenate for 5 minutes at room temperature to permit the complete dissociation of nucleoprotein complexes. Next, supplement the homogenate with 0.2 ml chloroform per 1 ml of TRI Reagent, cover the samples tightly and shake vigorously for 15 seconds. Store the resulting mixture at room temperature for 2-15 minutes and centrifuge at 12,000 g for 15 minutes at 4 C. Following centrifugation, the mixture separates into a lower red phenol-chloroform phase, interphase and the colorless upper aqueous phase. Transfer the aqueous phase to a fresh tube. Precipitate RNA from the aqueous phase by mixing with isopropanol. Use 0.5 ml of isopropanol per 1 ml of TRI Reagent used for the initial homogenization. Store samples at room temperature for 5-10 minutes and centrifuge at 12,000 g for 8 minutes at 4 - 25 C. Remove the supernatant and wash the RNA pellet ( by vortexing) with 75% ethanol and subsequent centrifugation at 7,500 g for 5 minutes at 4 - 25 C. Add at least 1 ml of 75% ethanol per 1 ml TRI Reagent used for the initial homogenization. Remove the ethanol wash and briefly air-dry the RNA pellet for 3 - 5 min. Dissolve RNA in DEPC-treated water by passing solution a few times through a pipette tip. Using an on-column DNase digestion with RNase-free DNase I (Qiagen) to purify the total RNA.
|
| Label |
biotin
|
| Label protocol |
Affymetrix Technical Manual, Prokaryotic Sample and Array Processing
|
| |
|
| Hybridization protocol |
The biotin-labeled target was hybridized to the Affymetrix GeneChip E. coli 2.0 array at 45ºC for 16 hour at 60 rpm using the Hybridization Oven 640 (Affymetrix), then a three-step fluorescent staining was conducted using the Fluidics Station 450 (Affymetrix) during the washing and staining procedure.
|
| Scan protocol |
The data were collected by using Affymetrix GeneChip Scanner 3000.
|
| Description |
RNA extracted from cells of E. coli K-12 JM109 with pMG1-IrrE after 1 M NaCl shock for 10 min
|
| Data processing |
Affymetrix GeneChip Operating Software (GCOS) Version 1.4 Details: Intra-chip normalizations were performed using Affymetrix Gene Chip Operating Software (GCOS). Default statistical parameters were used to normalize each chip to the same target intensity (1500) as described in the Affymetrix GeneChip Expression Analysis manual. All nine possible inter-chip comparisons were performed in GCOS. The data were subsequently exported to a Microsoft Excel spreadsheet for manipulation. Consensus “detection p-value”, “change p-value”, and “signal log ratios” were calculated, and the default E. coli array p-value cutoff parameters were applied to these consensus values to estimate the transcript change between two conditions and the transcript presence under each condition. Background-subtracted data sets were used to calculate up-regulated and down-regulated genes based on fold changes of greater than 2.
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| |
|
| Submission date |
Jul 31, 2009 |
| Last update date |
Jul 31, 2009 |
| Contact name |
Ming Chen |
| E-mail(s) |
chenmingbio@hotmail.com
|
| Organization name |
Biotechnology Research Institute, Chinese academy of Agricultural Sciences
|
| Street address |
Zhongguancun Nandajie 12th,Haidian Zone
|
| City |
Beijing |
| ZIP/Postal code |
100081 |
| Country |
China |
| |
|
| Platform ID |
GPL3154 |
| Series (1) |
| GSE17465 |
Transcriptional profile of Escherichia coli K12 strain JM109 harboring pMG1 and pMG1-IrrE under 1M NaCl shock |
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