|
| Status |
Public on Mar 03, 2014 |
| Title |
Δ2099 early stationary phase |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Δ2099 knockout at OD600 = 0.9688
|
| Organism |
Halobacterium salinarum NRC-1 |
| Characteristics |
strain: NRC-1
genotype: Δ2099
temperature: 37°C
od: 0.9688
|
| Treatment protocol |
Culture aliquots (2 ml) were collected over mid-log (OD600: ~0.4), late-log (OD600: ~0.8), early stationary, and late stationary phases of growth (as the carrying capacity differs for the two strains, stationary phase points were determined as occurring after an equivalent proportion of the duration of stationary phase.) Cells were centrifuged (16 000 g, 60 s) and flash frozen.
|
| Growth protocol |
The wild type (Dura3) and VNG2099C in-frame deletion strain (D2099) were batch cultured in flasks at 37°C with constant shaking (~220 rpm) in a complex media (CM: 250 g/l NaCl, 20 g/l MgSO4, 2 g/l KCl, 3 g/l sodium citrate, 10 g/l Oxoid brand bacteriological peptone). This standard CM was supplemented with 50mg/l uracil to compensate for the uracil deficiency caused by the Dura3 marker.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared from the cell pellets using the mirVANA RNA kit (Ambion, Austin, TX) according to manufacturer's instructions and then treated with RNase-free DNase (Promega).
|
| Label |
Cy3, Cy5
|
| Label protocol |
RNAs were direct labeled with Alexa547 and Alexa647 dyes (Kreatech). See Baliga NS, Bjork SJ, Bonneau R, Pan M, Iloanusi C, Kottemann MC, Hood L, DiRuggiero J. Systems level insights into the stress response to UV radiation in the halophilic archaeon Halobacterium NRC-1.Genome Res. 2004 Jun;14(6):1025-35.
|
| |
|
| Channel 2 |
| Source name |
Halobacterium salinarum NRC-1 reference sample
|
| Organism |
Halobacterium salinarum NRC-1 |
| Characteristics |
strain: NRC-1
genotype: wild type
temperature: 37°C
od: 0.5-0.6
|
| Treatment protocol |
Culture aliquots (2 ml) were collected over mid-log (OD600: ~0.4), late-log (OD600: ~0.8), early stationary, and late stationary phases of growth (as the carrying capacity differs for the two strains, stationary phase points were determined as occurring after an equivalent proportion of the duration of stationary phase.) Cells were centrifuged (16 000 g, 60 s) and flash frozen.
|
| Growth protocol |
The wild type (Dura3) and VNG2099C in-frame deletion strain (D2099) were batch cultured in flasks at 37°C with constant shaking (~220 rpm) in a complex media (CM: 250 g/l NaCl, 20 g/l MgSO4, 2 g/l KCl, 3 g/l sodium citrate, 10 g/l Oxoid brand bacteriological peptone). This standard CM was supplemented with 50mg/l uracil to compensate for the uracil deficiency caused by the Dura3 marker.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared from the cell pellets using the mirVANA RNA kit (Ambion, Austin, TX) according to manufacturer's instructions and then treated with RNase-free DNase (Promega).
|
| Label |
Cy5, Cy3
|
| Label protocol |
RNAs were direct labeled with Alexa547 and Alexa647 dyes (Kreatech). See Baliga NS, Bjork SJ, Bonneau R, Pan M, Iloanusi C, Kottemann MC, Hood L, DiRuggiero J. Systems level insights into the stress response to UV radiation in the halophilic archaeon Halobacterium NRC-1.Genome Res. 2004 Jun;14(6):1025-35.
|
| |
|
| |
| Hybridization protocol |
Whole-genome tiling arrays were designed using e-Array (Agilent Technologies) for the H. salinarum NRC-1 main chromosome (NC_002607), and pNRC100 (NC_001869) and pNRC200 (NC_002608) plasmids using a 60k feature design of 60-mer strand-specific probes spaced at 24 bp. Arrays included manufacturer’s control probes and were printed by Agilent Technologies. Hybridization and washing was performed as described (Turkarslan et al 2011), with dye flip experiments performed for each sample. See http://www.chem.agilent.com/Scripts/PDS.asp?lPage=34519 for details.
|
| Scan protocol |
Arrays were scanned in ScanArray 5000 (Perkin Elmer) using Packard BioChip ScanArray software. See http://www.systemsbiology.org/Scientists_and_Research/Technology/ISB_Facilities/Microarray for microarray scanning details.
|
| Description |
Normalized log10 intensity of X_INT/Y_INT (sample/reference) averaged over dye flip between sample and reference RNA.
|
| Data processing |
Spot finding was performed using Feature Extraction (Agilent Technologies). See http://www.systemsbiology.org/Scientists_and_Research/Technology/Data_Management/Microarray_Pipeline for more details.
Normalization and statistical analysis were performed as described before (Koide et al, 2009). Values are normalized log10 ratios representing test/reference. Interarray comparisons were normalized relative to a reference RNA sample generated from unperturbed H. salinarum NRC-1 batch culture in rich medium at OD600 0.5-0.6.
|
| |
|
| Submission date |
Apr 11, 2013 |
| Last update date |
Mar 03, 2014 |
| Contact name |
Elisabeth Wurtmann |
| Organization name |
Institute for Systems Biology
|
| Street address |
401 Terry Ave N
|
| City |
Seattle |
| State/province |
Washington |
| ZIP/Postal code |
98109 |
| Country |
USA |
| |
|
| Platform ID |
GPL17005 |
| Series (1) |
| GSE45988 |
H. salinarum NRC-1 vs VNG2099C knockout |
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