|
| Status |
Public on May 26, 2016 |
| Title |
ICL104_Mock_0hr_RNA_3 |
| Sample type |
RNA |
| |
|
| Source name |
Calu-3_mock-inoculated_0hr
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: Calu-3
virus: Mock
time: 0hr
biological_replicate: 3
|
| Treatment protocol |
Cells were infected with an MOI of 3. After infection, wash and replace with 2 ml of DMEM-F12 + .3% BSA and 1 ug/ml TPCK-trypsin.
|
| Growth protocol |
SCL002.0P: Maintenance, Plating, and Virus Infection of Calu-3 cells
|
| Extracted molecule |
total RNA |
| Extraction protocol |
TCL001.0P: Preparation of Samples from Calu-3 cells for Isolation of RNA
|
| Label |
Cy3
|
| Label protocol |
Total RNA was labeled according to Agilent’s Quick Amp Labeling protocol as part of the Agilent One-Color Microarray-Based Gene Expression Analysis Protocol (Agilent Technology). Total RNA from each sample was linearly amplified and labeled with Cy3-UTP.
|
| |
|
| Hybridization protocol |
Hybridization was performed according to Agilent One-Color Microarray-Based Gene Expression Analysis Protocol (Agilent Technology). The labeled cRNAs were purified by RNeasy Mini Kit (Qiagen). The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000. 1 μg of each labeled cRNA was fragmented by adding 11 μl 10 × Blocking Agent and 2.2 μl of 25×Fragmentation Buffer, then heated at 60 °C for 30 min, and finally 55 μl 2 × GE Hybridization buffer was added to dilute the labeled cRNA. 100μl of hybridization solution was dispensed into the gasket slide and assembled to the gene expression microarray slide. The slides were incubated for 17 hours at 65°C in an Agilent Hybridization Oven.
|
| Scan protocol |
The hybridized arrays were washed, fixed and scanned with using the Agilent DNA Microarray Scanner (part number G2505C).
|
| Data processing |
Scanned images were analyzed using Agilent Feature Extraction Software (v11.0.1.1). The limma package for R (available on Bioconductor) was used to perform background correction, quantile normalization (normalizeBetweenArrays), and summarization (avereps) to derive a single normalized intensity value per probe. Outlier samples were detected using PCA and by visual inspection of heatmaps, and all data was re-processed after removing outlier samples. All data processing for each of the biological replicates was performed independently of the other.
|
| |
|
| Submission date |
Apr 26, 2016 |
| Last update date |
Aug 31, 2016 |
| Contact name |
Natalie Heller |
| E-mail(s) |
natalie.heller@pnnl.gov
|
| Organization name |
PNNL
|
| Street address |
902 Battelle Blvd.
|
| City |
Richland |
| ZIP/Postal code |
99354 |
| Country |
USA |
| |
|
| Platform ID |
GPL13497 |
| Series (2) |
| GSE65575 |
Modeling Host Responses to Understand Severe Human Virus Infections |
| GSE80697 |
Human Calu-3 cell transcriptome response to wild-type pandemic H1N1 (A/California/04/2009), natural isolate [mRNA] |
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