|
| Status |
Public on Dec 02, 2015 |
| Title |
Mtb_2hr_normal pH_2 |
| Sample type |
RNA |
| |
|
| Source name |
Mtb H37Rv,2hrs,normal pH, replicate 2
|
| Organism |
Mycobacterium tuberculosis H37Rv |
| Characteristics |
treatment: normal pH
strain: Wild type strain of Mtb H37Rv
growth phase: early log phase
|
| Treatment protocol |
Early log phase culture cells were taken. Mtb and MtbΔwhiB3 were treated with 7H9-Ty--ADS medium of pH 4.5. Control Mtb cultures were treated with 7H9-Ty-ADS medium
|
| Growth protocol |
Mtb and MtbΔwhiB3 were seeded at 0.05 OD600 in 7H9-ADS medium and grown till early log phase of ~0.3.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Gene expression from Treated cultures were fixed using Guanidine thiocyanate, pelleted and total RNA was isolated using FastRNA PRO blue kit (according to manufacturer's instructions). RNA was purified using Qiagen RNeasy plus kit according to manufacturer's instructions. DNAse treatment was done using Ambion TURBO DNA-free kit according to manufacturer's instructions. RNA quality was measured using Agilent Bioanalyzer.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 50 ng RNA using the One-Color Low RNA Input Quick Amplification Labelling kit (Agilent Technologies, G4140-90040) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
The Gene Expression Hybridization Kit (Agilent Technologies, 5188-5279) and associated protocols were used (G4140-90040). The purified cRNA (600ng) was fragmented with fragmentation blocking mix by incubating samples at 60°C for 30 minutes. Microarrays were prepared in Agilent Technologies Hybridization Chamber according to manufacturers instructions (G2534-90001). Once loaded into thehybridization chamber, samples were placed in thehybridization oven (Agilent Technologies, G2505-80085) and incubated for 17 hours at 65°C while rotating at setting 10. Following hybridization, samples were washed according to procedure described by Agilent Technologies.
|
| Scan protocol |
Microarray slides were scanned at the resolution of 5µm using Agilent Microarray scanner (G2565CA) with scan control software as per manufacturer's instructions (G2505-90020). Settings included Agilent HD_GX_1colour (61X21.6mm), TIFF 20 bit, and Photomultiplier tube (PMT) gain 100%. Scanned image displays were analyzed by quantifying the pixel density of each hybridization spot using the Agilent Feature Extraction software (v10.5). Local background signals were subtracted from the data automatically by this software and raw data was obtained.
|
| Description |
Gene expression of Mtb early log phase cultures in 7H9-ADS medium
|
| Data processing |
Normalization was done to 50th percentile of each sample and data transformation was done to median of all samples using GeneSpring Gx V 12.0 from Agilent Technologies
|
| |
|
| Submission date |
Sep 19, 2014 |
| Last update date |
Dec 02, 2015 |
| Contact name |
Amit Singh |
| E-mail(s) |
[email protected]
|
| Phone |
9481700318
|
| Organization name |
Indian Institute of Science
|
| Department |
MCBL
|
| Street address |
CV Raman Road
|
| City |
Bangalore |
| State/province |
Karnataka |
| ZIP/Postal code |
560012 |
| Country |
India |
| |
|
| Platform ID |
GPL16972 |
| Series (1) |
| GSE61579 |
Transcriptomic analysis of Mtb H37Rv and MtbΔwhiB3 at pH 4.5 |
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