|
| Status |
Public on May 24, 2014 |
| Title |
48h Rach model, H37Rv vs. lsr2 mutant, replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Wild type H37Rv, 48h Rach dormancy model
|
| Organism |
Mycobacterium tuberculosis |
| Characteristics |
strain/background: H37Rv
genotype/variation: wild type
growth condition: 48h Rach dormancy model
|
| Treatment protocol |
For H2O2 experiments, 3mM H2O2 was added either to the aerobic samples or Rach model samples, and samples were harvested after 1h of treatment.
|
| Growth protocol |
Samples were grown in 50 mL volumes in 125 mL flasks. Samples for Rach models were grown in 16 mL cultures at an OD600 of 0.15 were started in glass tubes (16 mm X 125 mm) containing stir bars (12 mm X 4.5 mm). Minimal head space remained in the tubes (~1 mL). The tubes were sealed with caps containing butyl rubber septa and stirred at 60% of maximum speed using a rotary magnetic tumble stirrer from V & P Scientific (San Diego, CA).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cell pellets were flash frozen on dry ice, resuspended in Trizol, and bead-beaten 3 x 30 seconds. Resulting cell lysate was added to chloroform and nucleic acids were then precipitated in isopropanol, rehydrated in 75% ethanol, then DNase treated. Fur further purification, Qiagen RNeasy kit was used according to the manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
1.5 ug of RNA was primed with N6 primers, heated for 10 minutes at 65 C, then incubated 10 minutes at 25 C followed by 90 minutes at 42 C. 5mM A,G,C and 0.2mM T dNTP mix was used.
|
| |
|
| Channel 2 |
| Source name |
H37Rv Δlsr2, 48h Rach dormancy model
|
| Organism |
Mycobacterium tuberculosis |
| Characteristics |
strain/background: H37Rv
genotype/variation: Δlsr2
growth condition: 48h Rach dormancy model
|
| Treatment protocol |
For H2O2 experiments, 3mM H2O2 was added either to the aerobic samples or Rach model samples, and samples were harvested after 1h of treatment.
|
| Growth protocol |
Samples were grown in 50 mL volumes in 125 mL flasks. Samples for Rach models were grown in 16 mL cultures at an OD600 of 0.15 were started in glass tubes (16 mm X 125 mm) containing stir bars (12 mm X 4.5 mm). Minimal head space remained in the tubes (~1 mL). The tubes were sealed with caps containing butyl rubber septa and stirred at 60% of maximum speed using a rotary magnetic tumble stirrer from V & P Scientific (San Diego, CA).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cell pellets were flash frozen on dry ice, resuspended in Trizol, and bead-beaten 3 x 30 seconds. Resulting cell lysate was added to chloroform and nucleic acids were then precipitated in isopropanol, rehydrated in 75% ethanol, then DNase treated. Fur further purification, Qiagen RNeasy kit was used according to the manufacturer's instructions.
|
| Label |
Cy5
|
| Label protocol |
1.5 ug of RNA was primed with N6 primers, heated for 10 minutes at 65 C, then incubated 10 minutes at 25 C followed by 90 minutes at 42 C. 5mM A,G,C and 0.2mM T dNTP mix was used.
|
| |
|
| |
| Hybridization protocol |
Oligoarray control targets and hybridization buffer (Agilent In Situ Hybridization Kit Plus) were added, and samples were applied to microarrays enclosed in Agilent SureHyb-enabled hybridization chambers. After hybridization, slides were washed sequentially.
|
| Scan protocol |
Microarrays were scanned using a GenePix 4000b scanner (Axon Instruments). The intensities of the two dyes at each spot were quantified using GenePix Pro 6.0 (Molecular Devices).
|
| Description |
Biological replicate 2 of 3.
|
| Data processing |
ArrayStar software was used for microarray analysis. Experiments performed in triplicate or quadruplicate were loaded into ArrayStar and normalized using RMA. Statistical significance was determined using the Students t-test.
|
| |
|
| Submission date |
May 23, 2014 |
| Last update date |
May 24, 2014 |
| Contact name |
Martin Voskuil |
| E-mail(s) |
[email protected]
|
| Organization name |
CU-AMC
|
| Department |
Microbiology
|
| Street address |
12800 E. 19th Ave. Mail Stop 8333 RC1-North Room P18-9115
|
| City |
Aurora |
| State/province |
CO |
| ZIP/Postal code |
80045 |
| Country |
USA |
| |
|
| Platform ID |
GPL15398 |
| Series (1) |
| GSE57948 |
Mycobacterium tuberculosis Lsr2 is a global transcriptional regulator required for adaptation to changing oxygen levels and virulence |
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