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Status |
Public on Apr 07, 2014 |
Title |
hip mutant HT1170, biological rep2 |
Sample type |
RNA |
|
|
Source name |
hip mutant HT1170
|
Organism |
Mycobacterium tuberculosis |
Characteristics |
genotype/variation: hip mutant growth stage: stationary phase cells
|
Treatment protocol |
Stationary phase cultures were treated with two volumes RNA protect (Qiagen) prior to total RNA extraction.
|
Growth protocol |
M. tuberculosis wild type strain (mc[2]6020) and hip mutants were grown in Middlebrook 7H9 broth (with supplements) at 37°C in a shaking incubator (200 rpm) to stationary phase (two weeks).
|
Extracted molecule |
total RNA |
Extraction protocol |
Cells were resuspended in RNA Pro (MP Biochemicals) and lysed by bead beating. 2. Total RNA was extracted with chloroform and preciptated with isopropanol.
|
Label |
biotin
|
Label protocol |
At room temp, prepared an IVT Master Mix in a nuclease-free tube. Master mix consisted of: IVT Biotin Label, IVT Labeling Buffer and IVT Enzyme Mix. 2. Mixed well by pipetting up and down several times. 3. Transfered 30 µL of IVT Master Mix to each (30 µL) double-stranded cDNA sample. Mixed thoroughly. 4. Incubated the IVT reaction for 4 or 16 hours at 40 °C in a thermal cycler
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Hybridization protocol |
Mixed fragmented and labeled RNA with Control Oligonucleotide B2, 20X Hybridization Controls, 2X Hybridization Mix DMSO and Nuclease-free Water. 2. Hyb on thermocycler for 5 mins at 99 degrees. 3. Incubateed the probe array filled with Pre-Hybridization Mix at 45 °C for 10 minutes with rotation. 4. Transfered the hybridization cocktail that has been heated at 99 °C, in Step 3, to a 45 °C heat block for 5 minutes. 5. Spun the hybridization cocktail at maximum speed in a microcentrifuge for 5 minutes to collect any insoluble material from the hybridization mixture. 6. Removed the array from the hybridization oven. Vented the array with a clean pipette tip and extract the Pre-Hybridization Mix from the array with a micropipettor. Refiledl the array with the appropriate volume of the clarified hybridization cocktail, avoiding any insoluble matter at the bottom of the tube. 7. Placed probe array into the hybridization oven, set to 45°C. 8. Rotated array at 60 rpm. 9. Hybridize for 16 hours
|
Scan protocol |
Equipment and Consumables Required for Washing and Staining Arrays: GeneChip® Fluidics Station 450, GeneChip Scanner 3000 series, Affymetrix GeneChip® Command Console AGCC v1.1.
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Description |
Gene expression data from hip mutant strain stationary phase cells
|
Data processing |
Statistical analysis of the data was performed using R/Bioconductor with the oneChannelGUI package and comparing the twelve hip mutant strains to the wild type strain.
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Submission date |
Mar 06, 2014 |
Last update date |
Apr 07, 2014 |
Contact name |
Kim Lewis |
Organization name |
Northeastern University
|
Street address |
360 Huntington Ave
|
City |
Boston |
State/province |
MA |
ZIP/Postal code |
02115 |
Country |
USA |
|
|
Platform ID |
GPL17082 |
Series (1) |
GSE55647 |
Gene expression analysis of Mycobacterium tuberculosis high-persister (hip) mutants. |
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