|
| Status |
Public on Feb 16, 2018 |
| Title |
TAW5, Replicate 13 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
TAW5
|
| Organism |
Bacillus subtilis |
| Characteristics |
replication genotype: oriC- oriN+
dnaa, sda, and spo0a genotype: Pspank-dnaA
|
| Growth protocol |
Cells were grown to mid-log phase in batch culture at 37 °C in minimal media supplemented with 1% (w/v) arabinose, 0.5% (w/v) xylose, 0.1 mM IPTG, 40 µg/ml tryptophan, and 40 µg/ml phenylalanine.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cultures were fixed with an equal volume of -20C methanol. RNA was extracted using the Qiagen RNeasy kit according to the manufacturer's instructions.
|
| Label |
Cy5
|
| Label protocol |
10 µg of each RNA sample was reverse transcribed using Superscript II reverse transcriptase (Invitrogen), random hexamers, and aminoallyl-dUTP (Ambion). The cDNA was labeled by conjugation to monofunctional Cy3 or Cy5 dyes (Amersham) for reference or experimental samples, respectively. Each experimental sample was mixed with an aliquot of reference sample.
|
| |
|
| Channel 2 |
| Source name |
Reference RNA
|
| Organism |
Bacillus subtilis |
| Characteristics |
reference rna: Includes pooled total RNA from B. subtilis cultures grown in defined minimal medium and cultures treated with DNA-damaging agents, as described in PMID 19737352
|
| Growth protocol |
Cells were grown to mid-log phase in batch culture at 37 °C in minimal media supplemented with 1% (w/v) arabinose, 0.5% (w/v) xylose, 0.1 mM IPTG, 40 µg/ml tryptophan, and 40 µg/ml phenylalanine.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cultures were fixed with an equal volume of -20C methanol. RNA was extracted using the Qiagen RNeasy kit according to the manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
10 µg of each RNA sample was reverse transcribed using Superscript II reverse transcriptase (Invitrogen), random hexamers, and aminoallyl-dUTP (Ambion). The cDNA was labeled by conjugation to monofunctional Cy3 or Cy5 dyes (Amersham) for reference or experimental samples, respectively. Each experimental sample was mixed with an aliquot of reference sample.
|
| |
|
| |
| Hybridization protocol |
Salmon testes DNA and yeast tRNA were added, and each cDNA sample was hybridized to a DNA microarray at 42 °C overnight.
|
| Scan protocol |
Scanned on a GenePix 4000B scanner. Images were quantified using GenePix 3.0 (Axon Instruments).
|
| Data processing |
Loess normalized and background subtracted using the R statistical software package Linear Models for Microarray Data (LIMMA). [RGbkgd <- backgroundCorrect(RG, method=normexp, offset=50); normalizeWithinArrays(RGbkgd)]
|
| |
|
| Submission date |
Jul 14, 2016 |
| Last update date |
Feb 16, 2018 |
| Contact name |
Alan Grossman |
| E-mail(s) |
adg@mit.edu
|
| Organization name |
MIT
|
| Department |
Biology
|
| Street address |
31 Ames St.
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
| |
|
| Platform ID |
GPL10707 |
| Series (1) |
| GSE84421 |
Expression profiles of Bacillus subtilis strains deleted for or ectopically expressing dnaA, sda, and spo0A during exponential growth and after treatment with HPUra. |
|
