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Sample GSM441958 Query DataSets for GSM441958
Status Public on Aug 20, 2009
Title Third WT mouse vs third iNOS-/- mouse after 3 weeks infection (Replicate 3 dye-swap)
Sample type genomic
 
Channel 1
Source name Transposon library recovered from third WT C57BL/6J mouse after 3 weeks of infection
Organism Mycobacterium tuberculosis
Characteristics strain: H37Rv
source: Transposon library
Treatment protocol 10^6 colony forming units of the transposon library were injected into the tail vein of C57BL/6J or iNOS-/- mice (backcrossed to a C57BL/6J background). Three or four weeks after infection, mice were sacrificed, spleens were harvested, and bacteria were recovered from spleen homogenates by plating on 7H10 agar medium containing OADC.
Growth protocol A transposon library was made in the strain M. tuberculosis H37Rv using the MycoMarT7 phage. The transposon library was grow to mid-log phase, filtered through a 5 micron syringe filter, sonicated two times for 5 seconds each, diluted to approximately 10^7 colony forming units per ml.
Extracted molecule genomic DNA
Extraction protocol Genomic DNA was extracted using multiple steps including treatment with chloroform:methanol (2:1), lysozyme, SDS and proteinase K, extraction with phenol:chloroform, and precipitation with isopropanol and ethanol. Genomic DNA was then partially digested with MspI or HinP1I and ligated to adaptors. Transposon ends and adjacent genomic DNA were amplified using PCR, then an outward facing T7 promoter within the transposon was used to transcribe RNA. cDNA was made from the transcription products using aminoallyl-dUTP.
Label Cy5
Label protocol Cy3 or Cy5 were used to label aminoallyl-dUTPs within the cDNA.
 
Channel 2
Source name Transposon library recovered from third iNOS-/- mouse after 3 weeks of infection
Organism Mycobacterium tuberculosis
Characteristics strain: H37Rv
source: Transposon library
Treatment protocol 10^6 colony forming units of the transposon library were injected into the tail vein of C57BL/6J or iNOS-/- mice (backcrossed to a C57BL/6J background). Three or four weeks after infection, mice were sacrificed, spleens were harvested, and bacteria were recovered from spleen homogenates by plating on 7H10 agar medium containing OADC.
Growth protocol A transposon library was made in the strain M. tuberculosis H37Rv using the MycoMarT7 phage. The transposon library was grow to mid-log phase, filtered through a 5 micron syringe filter, sonicated two times for 5 seconds each, diluted to approximately 10^7 colony forming units per ml.
Extracted molecule genomic DNA
Extraction protocol Genomic DNA was extracted using multiple steps including treatment with chloroform:methanol (2:1), lysozyme, SDS and proteinase K, extraction with phenol:chloroform, and precipitation with isopropanol and ethanol. Genomic DNA was then partially digested with MspI or HinP1I and ligated to adaptors. Transposon ends and adjacent genomic DNA were amplified using PCR, then an outward facing T7 promoter within the transposon was used to transcribe RNA. cDNA was made from the transcription products using aminoallyl-dUTP.
Label Cy3
Label protocol Cy3 or Cy5 were used to label aminoallyl-dUTPs within the cDNA.
 
 
Hybridization protocol The Tecan HS400 hybridization station was used. Slides were washed with 5x SSC and 0.1% SDS at 42ºC for 30 s and allowed to soak in wash for 30 s. Prehybridization buffer was added at 42ºC for 30 min with agitation every 7 min for 1.5 min. Slides were washed twice with water for 30 s and soaked for 30 s each time. Slides were washed with 5x SSC and 0.1% SDS for 30 s and allowed to soak for 30 s at 23ºC. 50 pmol dye of each labeled cDNA suspended in hybridization buffer was injected into the slide chamber at 60ºC. Slides were heated to 95ºC for 2 min and hybridized at 42ºC for 16 h with 1.5 min agitation every 7 min. Slides were washed with 5x SSC and 0.1% SDS for 30 s and soaked for 30 s at 23ºC, washed with 0.2x SSC and 0.1% SDS for 30 s and soaked for 30 s at 23ºC, washed five times with 0.05x SSC for 30 s and soaked for 30 s each time at 23ºC, washed with 0.05x SSC for 30 s and soaked for 30 s at 23ºC, and dried at 30ºC for 90 min.
Scan protocol Scanned on a Genepix4000 scanner.
Description cDNA amplified from genomic DNA adjacent to transposon insertion sites
Biological replicate 3 of 5, dye swap
Data processing LOWESS normalized, background subtracted data, with values less than 0.01 set to 0.01, obtained from log2 of processed Red signal/processed Green signal or from log2 of processed Green signal/processed Red signal for dye-swaps. Genespring software was used.
 
Submission date Aug 18, 2009
Last update date Aug 19, 2009
Contact name Jeffrey P. Murry
E-mail(s) jmurry@salk.edu
Organization name Salk Institute
Department IMPL-Y
Street address 10010 N. Torrey Pines Road
City La Jolla
State/province CA
ZIP/Postal code 92037-1099
Country USA
 
Platform ID GPL9051
Series (1)
GSE17706 Mycobacterium tuberculosis TraSH experiment: Growth in wild type C57BL/6J versus iNOS-/- mice

Data table header descriptions
ID_REF
VALUE normalized log2 ratio (Cy5/Cy3) representing test/reference (iNOS/WT) or, for dye-swap technical replicates, normalized log2 ratio (Cy3/Cy5) representing test/reference (iNOS/WT)

Data table
ID_REF VALUE
Rv3464 -0.3997
Rv3463 0.1570
Rv1920 -0.5500
Rv3469c -0.3622
Rv3467 0.3528
Rv3466 -0.3383
Rv3454 2.6704
Rv1926c -0.0233
Rv3444c 0.2004
Rv3440c -1.8009
Rv3453 1.9553
Rv1924c -0.6712
Rv1925 -0.6873
Rv3439c 1.8742
Rv0872c 0.4489
Rv3483c 0.2666
Rv3482c -0.8211
Rv3487c 1.5499
Rv3486 -0.7710
Rv3485c -1.1649

Total number of rows: 2264

Table truncated, full table size 33 Kbytes.




Supplementary file Size Download File type/resource
GSM441958_3.3B.gpr.gz 840.6 Kb (ftp)(http) GPR
Processed data included within Sample table

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