|
| Status |
Public on Feb 13, 2018 |
| Title |
Anti-AT rep3 |
| Sample type |
RNA |
| |
|
| Source name |
Alveolar macrophages 24h post S. aureus infection, with AT neutralization
|
| Organism |
Mus musculus |
| Characteristics |
strain/background: C57BL/6J
gender: Female
age: 7 weeks
treatment: Anti-alpha toxin
infection: Staphylococcus aureus
cell type: Alveolar macrophage
|
| Treatment protocol |
Specific-pathogen-free 7- to 8-week-old female C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME) were briefly anesthetized and maintained in 3% isoflurane (Butler Schein™ Animal Health) with oxygen at 3 L/min and infected intranasally (S. aureus USA300 strain SF8300). All bacterial suspensions were administered in 50 µL of PBS. MEDI4893* (LC10) or c-IgG was administered in 0.5 mL intraperitoneally (IP) 24h prior to infection.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Alveolar macrophages were purified from the BAL of naïve and infected mice by CD11c bead selection (Stem Cell). Cells purified from 10 mice were pooled and treated as a single sample for analysis. Total RNA was extracted using RNeasy Mini Kit (Qiagen) following the manufacturer’s protocol and treated with RNase-free DNase I to remove genomic DNA contamination. RNA was quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) and the quality of RNA was assessed using the Agilent RNA ScreenTape assay in conjunction with a 4200 TapeStation system (Agilent Technologies). Only high-quality RNA samples with an RNA Integrity Number (RIN) greater than 9 were used for microarray hybridization.
|
| Label |
biotin
|
| Label protocol |
Briefly, 75 ng of total RNA was reverse transcribed to first strand cDNA with T7-oligo(dT) primer using ArrayScript reverse transcriptase, followed by second strand cDNA synthesis to generate double-stranded cDNA (ds-cDNA). Subsequently, the ds-DNA was used as a template for in vitro transcription to synthesize biotin-labeled antisense-RNA (aRNA) molecules.
|
| |
|
| Hybridization protocol |
The biotin-labeled aRNA was purified with RNA binding beads and then fragmented at 94°C for 35 min in fragmentation buffer (40 mM Tris-acetate, pH 8.2, 100 mM Potassium Acetate and 30 mM Magnesium Acetate). Fragmented aRNA (10 µg) was hybridized to Affymetrix GeneChip Mouse Genome 430 2.0 Array (Thermo Fisher Scientific) at 45 °C for 18 hrs.
|
| Scan protocol |
Affymetrix GeneChip Fluidics Station 450 was used for washing and staining of the arrays, and hybridized arrays were scanned using a GeneChip Scanner 300 7G (Thermo Fisher Scientific) according to the manufacturer’s user guide.
|
| Description |
RE726_LC10_3
|
| Data processing |
Array files were normalized using the RMA method, and analyzed with Limma for differential expression analysis and GAGE for gene set (pathway) enrichment.
|
| |
|
| Submission date |
Aug 09, 2017 |
| Last update date |
Feb 13, 2018 |
| Contact name |
Taylor Cohen |
| E-mail(s) |
[email protected]
|
| Phone |
3013982405
|
| Organization name |
MedImmune
|
| Street address |
One Medimmune Way
|
| City |
Gaithersburg |
| State/province |
MD |
| ZIP/Postal code |
20878 |
| Country |
USA |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE102444 |
Staphylococcus aureus evades macrophage killing through NLRP3 dependent effects on mitochondrial trafficking |
|
