|
| Status |
Public on Jun 22, 2013 |
| Title |
M1, NS1 F103L/M106I 3 |
| Sample type |
RNA |
| |
|
| Source name |
M1, NS1 F103L/M106I
|
| Organism |
Mus musculus |
| Characteristics |
cell line: M1
cell type: immortal kidney epithilium
|
| Treatment protocol |
Confluent monolayers of 1.5E6 M1 cells in 35 mm dishes were infected with rHK-wt or rHK NS mutant viruses (MOI = 2) or mock-infected with PBS in triplicate for n=3 biological replicates before overflay with 3 ml of MEM.
|
| Growth protocol |
M1 cells (Mouse kidney epithelium; ATCC, CRL-2038) were maintained in minimum essential medium (MEM). MEM was supplemented with L-glutamine (2 mM), Penicillin (100 U/ml), Streptomycin (100 ug/ml) and fetal bovine serum (FBS) (10%). All cells were incubated at 37°C in the presence of 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
At 8 hpi, the cellular RNA was collected using manufacturer’s protocol (QIAGEN RNeasy mini-kit) quantified using a PowerWave XS2, Microplate Spectrophotometer (BioTek Instruments, Inc. Winooski, VT, United States).
|
| Label |
Biotin
|
| Label protocol |
Sense strand target cDNA were prepared using the Ambion WT Expression kit (Life Technologies cat# 4411973/4) from 300 ng of total RNA. Fragmentation and labeling were performed according to the standard Affymetrix protocol from 5.5 ug of cDNA (Genechip WT Terminal Labeling and Hybridization Manual Rev.7, Affymetrix).
|
| |
|
| Hybridization protocol |
Target cDNA were hybridized to the Mouse Gene 1.0 ST array according to Affymetrix standard protocol (Genechip WT Terminal Labeling and Hybridization Manual Rev.7, Affymetrix). GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
|
| Scan protocol |
GeneChips were scanned using the Affymetrix Genechip Scanner 3000 7G.
|
| Description |
Affy#4486_1.0: biological replicate 3
|
| Data processing |
RMA normalization and scaling of data was performed with Affymetrix Expression Console v. 1.2
|
| |
|
| Submission date |
Jun 21, 2013 |
| Last update date |
Jun 22, 2013 |
| Contact name |
Earl Garnet Brown |
| E-mail(s) |
[email protected]
|
| Phone |
613 224 7533
|
| Organization name |
University of Ottawa
|
| Department |
Biochemistry Microbiology and Immunology
|
| Lab |
RGN 4109
|
| Street address |
451 Smyth Rd
|
| City |
Ottawa |
| State/province |
Ontario |
| ZIP/Postal code |
KIH 8M5 |
| Country |
Canada |
| |
|
| Platform ID |
GPL6246 |
| Series (1) |
| GSE48200 |
Influenza A/Hong Kong/156/1997(H5N1) virus NS1 gene mutations F103L and M106I both increase IFN antagonism, virulence and cytoplasmic localization but differ in binding to RIG-I and CPSF30 |
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