|
| Status |
Public on Sep 20, 2017 |
| Title |
WT vs cyc- stationary growth phase |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Wildtype_Statphase
|
| Organism |
Halobacterium salinarum |
| Characteristics |
strain: R1
genotype: wildtype
growth phase: stationary
|
| Treatment protocol |
After cell lysis and DNAase treatment (Invitrogen), RNA was extracted using spin columns (Agilent).
|
| Growth protocol |
H. salinarum WT and KO strains were cultured, in triplicate to minimize biological noise, under normal conditions with aeration (200 r.p.m.) in complete medium at 40°C (Oesterhelt & Krippahl (1983). Cultures were grown until logarithmic (OD600 = 0.3) and also until stationary phase (OD600 = 1.3) of growth.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
6 ug high quality total RNA was combined with 500 ng of random hexamers (Qiagen) and reverse transcribed to Cy3- or Cy5-dCTP (Amersham Pharmacia)-labeled cDNA using SuperScript II RNase H_ reverse transcriptase (Invitrogen).
|
| Label |
Cy3
|
| Label protocol |
Labeled cDNA targets of the WT and KO strains were mixed 1:1 and were mixed with hybridization buffer (Agilent) and hybridized to microarray slides, which were assembled into a hybridization chamber (Agilent) for 17 h at 65°C in the dark.
|
| |
|
| Channel 2 |
| Source name |
Knockout_Statphase
|
| Organism |
Halobacterium salinarum |
| Characteristics |
strain: NRC-1
genotype: cyc knockout
growth phase: stationary
|
| Treatment protocol |
After cell lysis and DNAase treatment (Invitrogen), RNA was extracted using spin columns (Agilent).
|
| Growth protocol |
H. salinarum WT and KO strains were cultured, in triplicate to minimize biological noise, under normal conditions with aeration (200 r.p.m.) in complete medium at 40°C (Oesterhelt & Krippahl (1983). Cultures were grown until logarithmic (OD600 = 0.3) and also until stationary phase (OD600 = 1.3) of growth.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
6 ug high quality total RNA was combined with 500 ng of random hexamers (Qiagen) and reverse transcribed to Cy3- or Cy5-dCTP (Amersham Pharmacia)-labeled cDNA using SuperScript II RNase H_ reverse transcriptase (Invitrogen).
|
| Label |
Cy5
|
| Label protocol |
Labeled cDNA targets of the WT and KO strains were mixed 1:1 and were mixed with hybridization buffer (Agilent) and hybridized to microarray slides, which were assembled into a hybridization chamber (Agilent) for 17 h at 65°C in the dark.
|
| |
|
| |
| Hybridization protocol |
Scanned on an Agilent G2505C US22502585 scanner.
|
| Scan protocol |
Image processing was performed using Agilent Feature Extraction software. Data were analyzed using the Agilent microarray data analyzing software GeneSpring version 11.5.1.
|
| Description |
1:1 combined, labeled cDNA of the WT and KO strain cultured until stationary growth phase
|
| Data processing |
Agilent Feature Extraction Software (v 10.5.1.1) was used for background subtraction and LOWESS normalization. Analysis was performed in the lab of Shiladitya DasSarma in Baltimore, MD, USA.
|
| |
|
| Submission date |
Sep 19, 2017 |
| Last update date |
Jan 23, 2018 |
| Contact name |
Walter Joseph Muller |
| E-mail(s) |
[email protected]
|
| Organization name |
University of the Free State
|
| Department |
Biotechnology
|
| Lab |
Biocatalysis
|
| Street address |
Decan Street
|
| City |
Bloemfontein |
| State/province |
Free state |
| ZIP/Postal code |
9301 |
| Country |
South Africa |
| |
|
| Platform ID |
GPL15637 |
| Series (1) |
|
