|
| Status |
Public on Nov 30, 2016 |
| Title |
reference strain Δ6_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Bacillus subtilis Δ6 (deletion of prophages)
|
| Organism |
Bacillus subtilis subsp. subtilis str. 168 |
| Characteristics |
genome size: genome size of 3.89 Mb
|
| Growth protocol |
Cultures of the reference strain Δ6 and the deletion strains PG10 and PS38 were grown under vigorous agitation at 37 °C in LB medium supplemented with 0.5 % glucose until OD at 600 nm of 0.5.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were harvested and total RNA was isolated by the mechanical cell disruption/ acid phenol method (Eymann et al., 2002; PMID 11948165), followed by a DNase I treatment in order to eliminate residual genomic DNA and column
purification using the RNA Clean-Up kit (Norgen Biotech Corp.). RNA quality was checked by means of an Agilent 2100 Bioanalyzer (Agilent Technologies).
|
| Label |
Cy3
|
| Label protocol |
For cDNA synthesis, 5 µg of total RNA were mixed with random primers (FairPlay III Microarray Labeling Kit) and spike-ins (One-Color RNA Spike-In Kit, Agilent Technologies) and incubated at 70°C for 10 min followed by 5 min incubation
on ice. Then, first-strand Master Mix, Actinomycin D (final conc. 40 µg/ml) and AffinityScript HC Reverse Transcriptase were added. The reaction was incubated for 60 min at room temperature and for 60 min at 42°C. After RNA hydrolysis,
cDNA was precipitated overnight at -20°C. NHS-ester dye coupling (CyDye Mono-Reactive Dye, GE Healthcare) and purification of labeled cDNA were performed according to the FairPlay III protocol. cDNA and Cy-dye concentrations
were quantified by means of a NanoDrop spectrophotometer.
|
| |
|
| Hybridization protocol |
200 ng of Cy3-labeled cDNA were hybridized to a tiled microarray (tiling step of 22 nucleotides) following Agilent’s hybridization, washing and scanning protocol (One-Color Microarray-based Gene Expression Analysis, version 5.5).
|
| Scan protocol |
The microarray was scanned using an Agilent Microarray Scanner G2505C (Agilent Technologies).
|
| Data processing |
Probe intensities were computed from the raw intensity data using a model of signal shift and drift and correcting for probe affinity variations as described before (Nicolas et al., 2009; PMID 19561016). An aggregated expression value was
computed for each annotated CDS and previously identified RNA feature (Nicolas et al., 2012; PMID 22383849) as the median log2 intensity of probes lying entirely within the corresponding region. To control for possible cross-hybridization
artefacts, probe sequences were BLAST-aligned against the whole chromosome sequence and probes with a SeqS value above a 1.5 cut-off were discarded (Wei et al., 2008; PMID 18385155). Gene-level intensities were scaled based on
the intensity values of ten different in-vitro synthesized transcripts contained in the One Color RNA Spike-In kit.
|
| |
|
| Submission date |
Jun 03, 2016 |
| Last update date |
Nov 30, 2016 |
| Contact name |
Ulrike Mäder |
| E-mail(s) |
[email protected]
|
| Organization name |
University Medicine Greifswald
|
| Department |
Functional Genomics
|
| Street address |
F.-L.-Jahn-Str. 15A
|
| City |
Greifswald |
| ZIP/Postal code |
D-17489 |
| Country |
Germany |
| |
|
| Platform ID |
GPL21981 |
| Series (1) |
| GSE82249 |
Transcriptome analysis of genome-reduced Bacillus subtilis strains |
|
