|
| Status |
Public on Jan 05, 2015 |
| Title |
H37Rv_Mg2000_exp1 |
| Sample type |
RNA |
| |
|
| Source name |
Mtb_WT_2000uM Mg2+
|
| Organism |
Mycobacterium tuberculosis |
| Characteristics |
strain background: H37Rv
genotype/variation: wild type
mg2+: 2000uM
|
| Growth protocol |
Mtb was grown in standing cultures at 37C for 5 days in Sauton’s media containing 250 or 2000 μM MgCl2 and 0.05% Tween 80. Flasks were shaken for 5 hours prior to harvest.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cultures were mixed with an equal volume of GTC buffer containing guanidinium thiocyanate (4 M), sodium lauryl sulfate (0.5%), trisodium citrate (25 mM), and 2-mercaptoethanol (0.1 M) and pelleted by centrifugation. Bacterial RNA was isolated as previously described (Ehrt, S. et al. (2002). Immunology of Infection 169–180).
|
| Label |
Cy3
|
| Label protocol |
Cy3 labeled RNA was prepared from 100 ng RNA using a Low Input Quick Amp Labeling Kit (Agilent) according to manufacturer's instructions.
|
| |
|
| Hybridization protocol |
600ng of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 uL containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 uL of 2x Agilent Gene Expression hybridization buffer (Hi-RPM) was added to the fragmentation mixture and hybridized to a custom-designed Mtb microarray for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 5 minute at room temperature with GE Wash Buffer 1 (Agilent) and 5 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 8x15k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
SAMPLE 4
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 019943_D_20080412) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
May 03, 2014 |
| Last update date |
Jan 05, 2015 |
| Contact name |
Sabine Ehrt |
| E-mail(s) |
sae2004@med.cornell.edu
|
| Organization name |
Weill Cornell Medical College
|
| Department |
Microbiology & Immunology
|
| Street address |
413 East 69th Street
|
| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10021 |
| Country |
USA |
| |
|
| Platform ID |
GPL16177 |
| Series (1) |
| GSE57263 |
Comparison of gene expression of WT and PerM mutant M. tuberculosis H37Rv grown in high and reduced magnesium |
|
