|
| Status |
Public on Jul 03, 2013 |
| Title |
wdNHBE with KY/136 at 36hpi, biological rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Well-differentiated human lung epithelial cells, infected with KY/136 (pdmH1N1) for 36h
|
| Organism |
Homo sapiens |
| Characteristics |
infection: 2009 pandemic H1N1 Influenza virus
cell type: cultured primary human lung epithelial cells
|
| Treatment protocol |
Infection of wd-NHBE cells were conducted in serum-free growth media provided by manufacturer (MatTek). Wd-NHBE cells, cultured at the air-liquid interface, were washed two times with Dulbecco’s phosphate buffered saline (DPBS) to remove mucus accumulation. Virus stocks were diluted in DPBS and cells were infected at a multiplicity of infection (MOI) of 3.0 for 1 h at 37°C, 5% CO2. Cells were washed twice with DPBS to remove unbound virus. Basal medium was removed and replaced with complete medium. Over the time course of infection, the basal media was removed and apical layer washed twice with DPBS supplemented with 0.2% BSA (Sigma) and stored at -80 degrees celcius until use.
|
| Growth protocol |
well-differentiated cells (wd-NHBE) were purchased from MatTek Corporation (EpiAirway PC-12, Ashland, MA). Cells were maintained by incubating the 12-well formatted transwell inserts at the air-liquid interface at 37°C under 5% CO2. The basal surface was submerged in AIR 100 (MatTek) complete growth media, made up of DMEM , epidermal growth factors, 5 µg/ml gentamicin, 0.25 µg/ml amphotericin B, and phenol red, and was replaced every 24 h until used for experiments.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 100ng total RNA (Gene Chip 3' IVT Expression Kit User Manual, 2008, Affymetrix).
|
| |
|
| Hybridization protocol |
Following fragmentation, 12.5ug of aRNA were hybridized for 16 hr at 45C on GeneChip Human (HG_U133 plus 2.0) Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450/250.
|
| Scan protocol |
Scanning of chip using Affymetrix Command Console (version 3.1)
|
| Description |
Gene expression data from well-differentiated primary human lung bronchial epithelial cells infected with H1N1
|
| Data processing |
Prior to statistical analyses, raw data were processed by Plier Workflow normalization method using Gene Console software (Affymetrix, version 1.3.1).
|
| |
|
| Submission date |
Jul 02, 2013 |
| Last update date |
Jul 03, 2013 |
| Contact name |
Rachael Gerlach |
| E-mail(s) |
rmlask01@louisville.edu
|
| Organization name |
University of Louisville
|
| Department |
Microbiology and Immunology
|
| Lab |
Jonsson
|
| Street address |
505 S Hancock, CTR 626
|
| City |
Louisville |
| State/province |
KY |
| ZIP/Postal code |
40202 |
| Country |
USA |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE48466 |
Expression data from well-differentiated human bronchial epithelial cells infected with H1N1 Influenza isolates |
|
