|
| Status |
Public on May 12, 2016 |
| Title |
TrxB2-DUC_DTT__atc_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Mtb,TrxB2-DUC,DTT 2mM,atc 800 ng/ml, 24hr, replicate2
|
| Organism |
Mycobacterium tuberculosis H37Rv |
| Characteristics |
genotype: TrxB2-DUC
treatment: DTT 2mM, atc 800 ng/ml
|
| Treatment protocol |
Atc was added to TrxB2-DUC culture to a final concentration of 800ng/ml and DTT was added to a final concentration of 2mM when the culture OD reached 0.5~0.6. Control group was not treated.
|
| Growth protocol |
Mtb was grown in Middlebrook 7H9 medium with 0.2% glycerol, 0.05% Tween-80, 0.5% BSA, 0.2% dextrose, and 0.085% NaCl.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cultures were mixed with 1:1 ratio with GTC buffer containing guanidinium thiocyanate (4 M), sodium lauryl sulfate (0.5%), trisodium citrate (25 mM), and 2-mercaptoethanol (0.1 M) and pelleted by centrifugation. Bacterial RNA was isolated as previously described (Goodsmith, N. et al. PLoS Pathog 2015 Feb;11(2):e1004645).
|
| Label |
Cy3
|
| Label protocol |
100 ng RNA was used for labeling with a Low Input Quick Amp Labeling Kit (Agilent) according to manufacturer's instructions
|
| |
|
| Hybridization protocol |
The One Color Cy3-labeled samples were initially mix with fragmentation mix consisting of 10X blocking Agent, nuclease free water, and 25X fragmentation buffer and incubated at 60 °C for 30minutes to fragment the cRNA. After incubation, 2X Hybridization Buffer was added to each sample to stop fragmentation reaction. The samples were hybridized to a custom-designed Mtb microarray for 17 hours at 65°C in a rotating hybridization oven. At the end of 17 hours the slides were taken out, dissembled in GE Wash Buffer 1, transferred to rack with clean GE Wash buffer 1 and mixed for 1-2 min at room temperature. Then the rack containing the hybridized slides was transferred to prewarmed (37°C) Wash Buffer 2 and stirred for 1 min. Finally, the rack containing the washed hybridized slides was slowly taken out of dish and allowed to air dry for 10-15 seconds.
|
| Scan protocol |
Each 8X15 hybridized slide was placed in slide holder and covered with ozone-barrier slide cover. The slides were immediately scanned in Agilent Sure Scan Microarray B Scanner using Protocol for One-Color Microarray Gene Expression. Once scanning was complete, the data was extracted using Agilent feature extraction software version 9.5.1.
|
| Data processing |
The raw data were extracted with Feature Extraction using Agilent default analysis settings.
|
| |
|
| Submission date |
Mar 04, 2016 |
| Last update date |
May 12, 2016 |
| Contact name |
Kan Lin |
| E-mail(s) |
kal2025@med.cornell.edu
|
| Organization name |
Weill Cornell Medical College
|
| Department |
Microbiology and Immunology
|
| Lab |
Dr. Sabine Ehrt
|
| Street address |
413 East 69th Street
|
| City |
NEW YORK |
| State/province |
New York |
| ZIP/Postal code |
10021 |
| Country |
USA |
| |
|
| Platform ID |
GPL16177 |
| Series (1) |
| GSE78894 |
Comparison of gene expression in M. tuberculosis TrxB2-DUC mutant treated or not with atc and DTT |
|
