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Sample GSM252056 Query DataSets for GSM252056
Status Public on Nov 20, 2008
Title V. parahaemolyticus gDNA 3%M90.4%glucose at 37°C vs gDNA non-replicating
Sample type genomic
 
Channel 1
Source name Genomic DNA from non-replicating cells
Organism Vibrio parahaemolyticus RIMD 2210633
Characteristics Strain: RIMD2210633
Growth protocol To obtain non-replicating bacteria, cells were spread onto Luria-Bertani (LB) Agar followed by a 24 h incubation t 20ºC and a 24 h incubation at 4ºC. Other samples were grown at 37ºC in liquid LB with 3% NaCl or liquid M9 with 3% NaCl and 0.4% glucose until an OD600 of 0.2, 0.5 or 3.0 was reached.
Extracted molecule genomic DNA
Extraction protocol Genomic DNA was purified using the DNeasy Tissue Kit (Qiagen) according to the manufacturer's instruction. RNA was extracted using TRIzol Reagent (Invitrogen) and purified with RNeasy Mini Kit (Qiagen) following the manufacturers protocols.
Label Cy5
Label protocol For generation of aminoallyl-labelled product from gDNA, the general procedures accompanying BioPrime Array CGH Genomic Labelling System (Invitrogen) were followed with a few modifications. Genomic DNA (2 μg) was incubated for 5 min at 95ºC with 20 μl 2.5X random primer solution in 44 μl reactions. Mixtures were cooled on ice for 3 min before addition of 5 μl 10X dNTP-aminoallyl dUTP (5 mM dATP, dCTP and dGTP, 2 mM dTTP and 3 mM aadUTP, Sigma) and 1 μl Exo-Klenow polymerase (40U, Invitrogen) and a further incubation at 37ºC for 1 h. Reactions were cleaned, precipitated and washed. For generation of aminoallyl-labelled product from total RNA, reagents included in SuperScript™ III Reverse Transcriptase Kit (Invitrogen) were used. In brief, 20 μg RNA was mixed with 10 μg random hexamers in a 22 μl reaction that was incubated at 70ºC for 5 min before cooling on ice. Addition of 5X First-Strand Buffer (8 μl), 0.1 M DTT (2 μl), SuperScript™ III RT (4 μl) and 10X dNTP-aminoallyl dUTP (4 μl) was followed by a 3 h incubation at 46ºC. Samples were cleaned, precipitated and washed. Finally, aminoallyl-labelled nucleic acids were resuspended in 10 μl 50 mM NaCO3 (pH 9.0), and 10 μl of either Cy3 or Cy5 monofunctional reactive dye (GE Healthcare) was added. Mixtures were incubated in the dark for 1 h at room temperature and unincorporated dye was removed on CENTRI-SEP Columns (Princeton Separations) following the manufacturer’s instructions before precipitation and wash.
 
Channel 2
Source name Genomic DNA from cells grown at 37°C in M9 3% NaCl and 0.4% glucose harvested at OD600=0.5
Organism Vibrio parahaemolyticus RIMD 2210633
Characteristics Strain: RIMD2210633
Growth protocol To obtain non-replicating bacteria, cells were spread onto Luria-Bertani (LB) Agar followed by a 24 h incubation t 20ºC and a 24 h incubation at 4ºC. Other samples were grown at 37ºC in liquid LB with 3% NaCl or liquid M9 with 3% NaCl and 0.4% glucose until an OD600 of 0.2, 0.5 or 3.0 was reached.
Extracted molecule genomic DNA
Extraction protocol Genomic DNA was purified using the DNeasy Tissue Kit (Qiagen) according to the manufacturer's instruction. RNA was extracted using TRIzol Reagent (Invitrogen) and purified with RNeasy Mini Kit (Qiagen) following the manufacturers protocols.
Label Cy3
Label protocol For generation of aminoallyl-labelled product from gDNA, the general procedures accompanying BioPrime Array CGH Genomic Labelling System (Invitrogen) were followed with a few modifications. Genomic DNA (2 μg) was incubated for 5 min at 95ºC with 20 μl 2.5X random primer solution in 44 μl reactions. Mixtures were cooled on ice for 3 min before addition of 5 μl 10X dNTP-aminoallyl dUTP (5 mM dATP, dCTP and dGTP, 2 mM dTTP and 3 mM aadUTP, Sigma) and 1 μl Exo-Klenow polymerase (40U, Invitrogen) and a further incubation at 37ºC for 1 h. Reactions were cleaned, precipitated and washed. For generation of aminoallyl-labelled product from total RNA, reagents included in SuperScript™ III Reverse Transcriptase Kit (Invitrogen) were used. In brief, 20 μg RNA was mixed with 10 μg random hexamers in a 22 μl reaction that was incubated at 70ºC for 5 min before cooling on ice. Addition of 5X First-Strand Buffer (8 μl), 0.1 M DTT (2 μl), SuperScript™ III RT (4 μl) and 10X dNTP-aminoallyl dUTP (4 μl) was followed by a 3 h incubation at 46ºC. Samples were cleaned, precipitated and washed. Finally, aminoallyl-labelled nucleic acids were resuspended in 10 μl 50 mM NaCO3 (pH 9.0), and 10 μl of either Cy3 or Cy5 monofunctional reactive dye (GE Healthcare) was added. Mixtures were incubated in the dark for 1 h at room temperature and unincorporated dye was removed on CENTRI-SEP Columns (Princeton Separations) following the manufacturer’s instructions before precipitation and wash.
 
 
Hybridization protocol Cy3- or Cy5- labeled probes (21.6 µl each) were mixed and 18 μl 20X SSC (3 M NaCl, 0.3 M HOC(COONa)(CH2COONa)2), 7.2 μl human CotI DNA (Invitrogen) and 3.6 μl 10% SDS was added. Hybridisation mixtures were incubated at 96ºC for 5 min, chilled on ice and kept at 55ºC until application onto microarray slides. Slides were covered with a MAUI AO Lid and insertion into MAUI hybridization chambers (BioMicro Systems) before application of the pre-warmed hybridisation mixture and 16 h incubation at 55ºC followed. After hybridisation, slides were washed twice in 2X SSC, 0.1 % SDS at 55ºC for 10 min, twice in 0.2X SSC, 0.1 % SDS at room temperature for 10 min, twice in 0.2X SSC at room temperature for 10 min and finally in ethanol for 1 min.
Scan protocol Slides were scanned with a ScanArrayExpress scanner (PerkinElmer). Obtained data was analysed by ScanArrayExpress software version 3.0 (PerkinElmer).
Description none
Data processing LOWESS normalised, background subtracted data obtained from log2 of processed Cy3 signal/processed Cy5 signal.
 
Submission date Dec 19, 2007
Last update date Nov 19, 2008
Contact name Kaori Izutsu
Organization name Osaka University
Department Research Institute for Microbial Diseases
Lab Laboratory of Genomic Research on Pathogenic Bacteria
Street address 1-1 Yamadaoka
City Suita
State/province Osaka
ZIP/Postal code 565-0871
Country Japan
 
Platform ID GPL6058
Series (1)
GSE9968 Examination of growth mode dependent replication dynamics and expression levels for Vibrio parahaemolyticus RIMD2210633.

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

Data table
ID_REF VALUE
1 0.13
2 0.175
3 0.196
4 0.061
5 0.055
6 0.257
7 0.064
8 0.362
9
10 -0.047
11 0.426
12 0.059
13 -0.002
14 0.016
15 0
16 0.176
17 0.175
18 0.198
19 0.167
20 0.065

Total number of rows: 4866

Table truncated, full table size 52 Kbytes.




Supplementary file Size Download File type/resource
GSM252056.txt.gz 175.9 Kb (ftp)(http) TXT
Processed data included within Sample table

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