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Sample GSM2414902 Query DataSets for GSM2414902
Status Public on Apr 01, 2019
Title 8 hours at 2000 mM sucrose_replicate 2
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 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).
 
Channel 2
Source name Mtb_treated by 2000 mM sucrose
Organism Mycobacterium tuberculosis
Characteristics strain: H37Rv
treatment: bacteria 2000 mM sucrose 8hours
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).
 
 
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 2 out of 6 8hrs 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.
 
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)
GSE90839 Osmotic stress response in Mtb

Data table header descriptions
ID_REF
VALUE log2 ratio (Cy5/Cy3)

Data table
ID_REF VALUE
DarkCorner -0.55142313
BUGS0000000635874 -0.10826114
BUGS0000000572230 0.11475059
BUGS0000000568351 -1.719982
BUGS0000000570111 0.001297467
BUGS0000000580269 -0.8717149
BUGS0000000635301 -1.4219818
BUGS0000000572954 0.15785691
BUGS0000000633957 0.7377538
BUGS0000000573611 -0.118020594
BUGS0000000569441 -0.48051262
BUGS0000000577015 -0.17134202
BUGS0000000573235 0.6816128
BUGS0000000571725 -1.0458888
BUGS0000000568734 -0.42599112
BUGS0000000570908 -0.17156112
BUGS0000000578362 0.5061865
BUGS0000000571715 -0.8103778
BUGS0000000569113 0.19247091
BUGS0000000575760 -1.0286671

Total number of rows: 12920

Table truncated, full table size 365 Kbytes.




Supplementary file Size Download File type/resource
GSM2414902_TB_252754310066_S01_BUGS_107_Aug10_1_3.txt.gz 1.3 Mb (ftp)(http) TXT
Processed data included within Sample table

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