|
| Status |
Public on Jul 25, 2008 |
| Title |
C t=0 Cy3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Total RNA from S. solfataricus control cells labeled with Cyanine-3 (green) at t=0 h post infection.
|
| Organism |
Saccharolobus solfataricus |
| Characteristics |
Strain 2-2-12
t=0 h post infection
|
| Extracted molecule |
total RNA |
| Extraction protocol |
mirVana total RNA extraction (Ambion)
|
| Label |
Cy3
|
| Label protocol |
2-5 µg of total RNA were primed with 5 ug random primer (hexamer) at 70°C for 10 min, then reversed transcribed at 42°C for 1 h in the presence of 400 U SuperScript II RTase (Invitrogen), and 100 µM each dATP, dTTP, dGTP, with 20 µM dCTP, 80 µM aminoallyl-dUTP (Sigma), and DTT. RNA was then degraded with 200nM EDTA and 1M NaOH, and labeled with CyDye Reactive Dye, post labeling (GE Healthcare) and cDNAs were purified using MinElute Enzymatic Cleanup Kit (Qiagen).
|
| |
|
| Channel 2 |
| Source name |
S. solfataricus + STIV labeled with Cyanine-5 (red) at t=0 h post infection.
|
| Organism |
Saccharolobus solfataricus |
| Characteristics |
Strain 2-2-12
t=0 post infection
|
| Treatment protocol |
Infection of cells with STIV at an MOI of approx. 1.5.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
mirVana total RNA extraction (Ambion)
|
| Label |
Cy5
|
| Label protocol |
2-5 µg of total RNA were primed with 5 ug random primer (hexamer) at 70°C for 10 min, then reversed transcribed at 42°C for 1 h in the presence of 400 U SuperScript II RTase (Invitrogen), and 100 µM each dATP, dTTP, dGTP, with 20 µM dCTP, 80 µM aminoallyl-dUTP (Sigma), and DTT. RNA was then degraded with 200nM EDTA and 1M NaOH, and labeled with CyDye Reactive Dye, post labeling (GE Healthcare) and cDNAs were purified using MinElute Enzymatic Cleanup Kit (Qiagen).
|
| |
|
| |
| Hybridization protocol |
Samples were mixed (18 uL total) with 42.6 uL of hybridization mix (45% formamide, 5X SSC, 0.1% SDS) and applied to prehybridized (1% BSA, 5X SSC, 0.1% SDS 40 min followed by 3X washes in water and 1X wash in isopropanol) microarrays. After hybridization, slides were washed sequentially with 6x SSC/0.005% Triton X-102 and 0.1x SSC/0.005% Triton X-102 before scanning. Slides were hybridized for 20 h at 42°C in hybridization cassettes (Array-It), and washed 5 min, 2X SSC, 0.1% SDS; 20 min, 0.1X SSC, 0.1% SDS, 5X 0.1X SSC and dried.
|
| Scan protocol |
Scanned on an GenePix 4000B.
Images were quantified using GenePix Pro 6.0.
|
| Description |
Biological replicate 3 of 3.
|
| Data processing |
Background calculated and subtracted using morphological opening followed by closing in GenePix 6.0, LOWESS normalization in GenespringGX 7.3.
|
| |
|
| Submission date |
Jul 24, 2008 |
| Last update date |
Jul 24, 2008 |
| Contact name |
Kate McInnerney |
| E-mail(s) |
[email protected]
|
| Phone |
406-994-5666
|
| Fax |
406-994-4926
|
| URL |
http://cores.montana.edu
|
| Organization name |
Montana State University
|
| Department |
Microbiology
|
| Lab |
Functional Genomics Facility
|
| Street address |
109 Lewis Hall
|
| City |
Bozeman |
| State/province |
MT |
| ZIP/Postal code |
59717 |
| Country |
USA |
| |
|
| Platform ID |
GPL7093 |
| Series (1) |
| GSE12235 |
Transcriptome analysis of infection of the archaeon Sulfolobus solfataricus with STIV |
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