|
| Status |
Public on Apr 01, 2019 |
| Title |
2 hours at 2000 mM sucrose_replicate 5 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
MTb_control
|
| Organism |
Mycobacterium tuberculosis |
| Characteristics |
strain: H37Rv
treatment: bacteria 10 mM NaCl
|
| Treatment protocol |
Bacteria were stressed by addition of NaCl to a concentration of 250 mM or 1000 mM NaCl for 2, 8 and 24h at mid-log phase (light transmittance at 600 nm ∼ 0.6).
|
| Growth protocol |
M. tuberculosis was grown in 7H9 liquid medium in roller bottles at 37 °C containing 10mM NaCl.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using a Fast RNA Pro Blue kit (MP Biomedicals). RNA was treated with RNase-free DNase (Promega) and purified using RNAeasy columns (Qiagen) according to the manufacturer's instructions.
|
| Label |
cy5
|
| Label protocol |
Fluorescently labeled cDNA was generated from total RNA (1 μg) by direct incorporation of Cy3- or Cy5-dCTP (GE Healthcare) using Superscript II Reverse Transcriptase (Invitrogen Life Technologies) in the presence of random hexamers (3 μg), dNTPs (185 μM dCTP and 463 μM each of dATP, dGTP and dTTP) and Cy3-dCTP or Cy5-dCTP (1.7 nmoles). RNA and random hexamers were initially mixed, made up to a volume of 11 μl and heated to 95 °C for 5 min. The mixture was then chilled on ice for 2 min. Remaining components of the reverse transcriptase reaction were added and the mixture was incubated for 10 min at 25 °C, followed by 42 °C for 90 min. Samples to be compared were mixed and the labelled cDNA was purified using MinElute PCR purification columns (Qiagen).
|
| |
|
| Channel 2 |
| Source name |
Mtb_treated by 2000 mM sucrose
|
| Organism |
Mycobacterium tuberculosis |
| Characteristics |
strain: H37Rv
treatment: bacteria 2000 mM sucrose 2hours
|
| Treatment protocol |
Bacteria were stressed by addition of NaCl to a concentration of 250 mM or 1000 mM NaCl for 2, 8 and 24h at mid-log phase (light transmittance at 600 nm ∼ 0.6).
|
| Growth protocol |
M. tuberculosis was grown in 7H9 liquid medium in roller bottles at 37 °C containing 10mM NaCl.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using a Fast RNA Pro Blue kit (MP Biomedicals). RNA was treated with RNase-free DNase (Promega) and purified using RNAeasy columns (Qiagen) according to the manufacturer's instructions.
|
| Label |
cy3
|
| Label protocol |
Fluorescently labeled cDNA was generated from total RNA (1 μg) by direct incorporation of Cy3- or Cy5-dCTP (GE Healthcare) using Superscript II Reverse Transcriptase (Invitrogen Life Technologies) in the presence of random hexamers (3 μg), dNTPs (185 μM dCTP and 463 μM each of dATP, dGTP and dTTP) and Cy3-dCTP or Cy5-dCTP (1.7 nmoles). RNA and random hexamers were initially mixed, made up to a volume of 11 μl and heated to 95 °C for 5 min. The mixture was then chilled on ice for 2 min. Remaining components of the reverse transcriptase reaction were added and the mixture was incubated for 10 min at 25 °C, followed by 42 °C for 90 min. Samples to be compared were mixed and the labelled cDNA was purified using MinElute PCR purification columns (Qiagen).
|
| |
|
| |
| Hybridization protocol |
Mycobacterium tuberculosis whole genome microarray slides (prepared at St. George's, University of London) were initially prehybridized in 3.5× SSC, 0.1% SDS and 10 mg/mL BSA for 20 min at 65 °C and then washed with deionized water and isopropanol. Labelled samples were heated for 2 min at 95 °C in 4xSSC and 0.3% SDS and hybridized on microarray slides under Lifter Slips (Thermo Scientific) for 16–20 h at 65 °C in a dark hybridization chamber. Hybridized slides were washed once with 1XSSC, 0.05% SDS at 65 °C and then twice with 0.06xSSC at room temperature.
|
| Scan protocol |
Scan on Agilent Technologies Scanner G2505C
Grids were fitted to the raw microarray images, and background normalization and spot quantitation was performed using Bluefuse software (BlueGnome Ltd., Cambridge, United Kingdom) and normalized readings were plotted using GeneSpring 10 software (Silicon Genetics).
|
| Description |
Biological replicate 5 out of 6 2hrs 2000 mM sucrose
|
| Data processing |
Data were obtained for six slides, including dye swaps, from three bacterial cultures. Data were initially filtered on expression and the lower 20th percentile was eliminated from the analysis. Genes that showed >2-fold change in absolute expression with a p-value <0.05 (Student's t-test) were considered to be altered.
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| |
|
| Submission date |
Dec 03, 2016 |
| Last update date |
Apr 01, 2019 |
| Contact name |
larrouy-maumus gerald |
| E-mail(s) |
glarrouymaumus@gmail.com
|
| Phone |
07545633280
|
| Organization name |
Imperial college london
|
| Department |
life sciences
|
| Street address |
exhibition road
|
| City |
London |
| ZIP/Postal code |
sw7 2az |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL22732 |
| Series (1) |
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