|
| Status |
Public on Feb 09, 2017 |
| Title |
Edoardo_251914810017-3_2.txt #1 control |
| Sample type |
RNA |
| |
|
| Source name |
suis WT O.D. 0.04 + DNA 45’ (No peptide)
|
| Organism |
Streptococcus suis |
| Characteristics |
strain: P1/7
|
| Treatment protocol |
RNA was extracted at the start of the experiment and after 5, 10 and 45 minutes after induction of competence (t=0 for all conditions)
|
| Growth protocol |
S. suis was grown in Todd-Hewitt broth (THB) (Difco) at 37°C under 5% CO2 for 18 hr.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction was preceded by induction of competence as described in [7]. Briefly S. suis S10 was grown to OD600nm 0.04. Thirty five ml of culture was collected and donor DNA (pNZ8048, 350 µg) in EB buffer (10 mM Tris-Cl, pH 8.5) was added to the bacteria along with synthetic XIP (GNWGTWVEE) at a final concentration of 250 µM [6]. Control extracts were obtained using same quantities of culture with donor DNA added but without XIP stimulation. Ten ml of the induced culture were collected after 5, 15 and 45 minutes following XIP exposure. Ten ml of uninduced cultures were collected at 15 and 45 minutes, spun down and the pellets were resuspended in 2.5 mL PBS plus 5 mL RNAprotect buffer (Promega). After 5 minutes of incubation the bacteria were collected via centrifugation and immediately frozen in liquid nitrogen until further handling. The frozen pellet was dissolved in 110 µl of TE containing proteinase K and lysozymes (1.25 µg/mL and 15 ug/ml respectively) and incubated for 10 minutes. The bacterial pellet was dissolved in 700 µL RLT buffer (Promega) containing 7 µl of freshly added β-mercaptoethanol and the bacteria were disrupted using a FastPrep-24 (MP. Biomedicals, Solon, OH) for 20 sec at 6.0 m/sec. Total RNA was purified using the RNeasy Mini Kit (Qiagen). The quality and the concentration of RNA were assessed with an Experion System (Bio-Rad) and by analysis of the A260/A280 ratio (NanoDrop 8000 UV-Vis Spectrophotometer). cDNA was synthesized with a SuperScript III Reverse Transcriptase kit (Invitrogen) using Aminoallyl-dUTP as dUTPs source and purified with the Illustra CyScribe GFX Purification Kit (GE Healthcare). The cDNA was labelled with CyDye Post-Labeling Reactive Dye Pack (GE Healthcare).
|
| Label |
Cy5
|
| Label protocol |
RNA (1 μg) from S. suis samples was labeled using the Cyanine 5 (Cy5) labeling reaction (indirect labeling of synthesized cDNA with the CyScribe Post-Labeling and Purification kit; Amersham Biosciences, Buckinghamshire, United Kingdom).
|
| |
|
| Hybridization protocol |
Agilent hybridization protocol for Two-color microarray-based Gene expression analysis. Version 5.5
|
| Scan protocol |
Scanned on an ScanArray Express 4000 scanner (Perkin Elmer, Wellesley, MA). Images were quantified using ImaGene Version 7.5 software (BioDiscovery Inc., Marina Del Rey, CA, USA).
|
| Data processing |
scanned slides were Lowess normalised using Prep (van Hijum, Applied Bioinformatics 2003 2(4):241-244).
Background subtracted, Lowess normalised, Cy5/Cy3 ratios (no log transformation); interslide scaling was performed using PostPrep (van Hijum, Applied Bioinformatics 2003 2(4):241-244). The raw numbers from this analysis were multiplied by 10000 yielding the numbers that are now in the Matrix sheet.
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| |
|
| Submission date |
Oct 29, 2015 |
| Last update date |
Feb 09, 2017 |
| Contact name |
Peter van Baarlen |
| E-mail(s) |
peter.vanbaarlen@wur.nl
|
| Organization name |
Wageningen University
|
| Department |
Animal Sciences
|
| Lab |
Host-Microbe Interactomics
|
| Street address |
De Elst 1
|
| City |
Wageningen |
| ZIP/Postal code |
6708 WD |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL16024 |
| Series (1) |
| GSE74507 |
The dynamic transcriptome of natural competence in Streptococcus suis |
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