|
| Status |
Public on Jul 12, 2018 |
| Title |
CC036d3_4 |
| Sample type |
RNA |
| |
|
| Source name |
blood
|
| Organism |
Mus musculus |
| Characteristics |
strain: CC036
gender: female
age: 8-12 weeks
tissue: blood
infection status: infected
infection severity: severe
infection day: d3
|
| Treatment protocol |
The CC strains (CC001, CC002, CC003, CC004, CC005, CC006, CC019, CC036, CC041, CC051 and CC053) were obtained from the University of North Carolina (Chapel Hill, NC) and bred in our animal facility. All mice were maintained under specific pathogen-free conditions and according to the German animal welfare law. Female, 8- to 12-week-old mice were anesthetized by intra-peritoneal injection with Ketamine/Xylazine (100 mg/ml Ketamine and 20 mg/ml Xylazine) in sterile natrium chloride solution. The doses were adjusted to the individual body weight using 200 µl/20 g body weight. Mice were then intra-nasally infected with 20 µl virus solution (10 FFU). Body weight was monitored daily as percentage of initial weight at the day of the infection. Mice that lost more than 30% of their body weight had to be euthanized for ethical reasons and were scored as dead.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Eye blood was collected in RNAprotect animal blood tubes (Qiagen) and immediately stored at -20 °C for at least 5 days. Blood RNA was isolated with the RNeasy protect animal blood kit (Qiagen). RNA concentration was measured with the NanoDrop (Thermo Scientific).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer's instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.5 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities.
|
| |
|
| Submission date |
Feb 08, 2018 |
| Last update date |
Jul 12, 2018 |
| Contact name |
Robert Geffers |
| E-mail(s) |
robert.geffers@helmholtz-hzi.de
|
| Phone |
+49 531-6181-3058
|
| Organization name |
HCI - Helmholtz Centre for Infection Research
|
| Department |
Dep. Molecular Bacteriology
|
| Lab |
Genome Analytics
|
| Street address |
Inhoffenstr. 7
|
| City |
Braunschweig |
| ZIP/Postal code |
38124 |
| Country |
Germany |
| |
|
| Platform ID |
GPL11202 |
| Series (1) |
| GSE110384 |
The host response to influenza virus infection |
|
