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Sample GSM2414934 Query DataSets for GSM2414934
Status Public on Apr 01, 2019
Title 2 hours at 250 mM NaCl_replicate 4
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 250 mM NaCl
Organism Mycobacterium tuberculosis
Characteristics strain: H37Rv
treatment: bacteria 250 mM NaCl 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 4 out of 6 2hrs 250 mM NaCl
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 0.34943673
BUGS0000000635874 0.11066933
BUGS0000000572230 0.11642367
BUGS0000000568351 0.83336604
BUGS0000000570111 0.55590713
BUGS0000000580269 0.9918059
BUGS0000000635301 1.1432015
BUGS0000000572954 -0.30638325
BUGS0000000633957 -0.64479494
BUGS0000000573611 0.7982755
BUGS0000000569441 0.5076261
BUGS0000000577015 1.1674223
BUGS0000000573235 -0.2376895
BUGS0000000571725 0.9871274
BUGS0000000568734 0.5006155
BUGS0000000570908 0.7908148
BUGS0000000578362 -0.5441693
BUGS0000000571715 1.0204587
BUGS0000000569113 -0.65647745
BUGS0000000575760 1.0372478

Total number of rows: 12920

Table truncated, full table size 364 Kbytes.




Supplementary file Size Download File type/resource
GSM2414934_TB_252754310068_S01_BUGS_107_Aug10_2_2.txt.gz 1.3 Mb (ftp)(http) TXT
Processed data included within Sample table

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