|
| Status |
Public on Aug 20, 2009 |
| Title |
Second WT mouse vs second iNOS-/- mouse after 4 weeks infection (Replicate 2 dye-swap) |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Transposon library recovered from second WT C57BL/6J mouse after 4 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 second iNOS-/- mouse after 4 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 2 of 8, 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 |
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