|
| Status |
Public on Apr 01, 2023 |
| Title |
pig #2 macrophages infected with VSV NJ95COB |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
universal control
|
| Organism |
Sus scrofa |
| Characteristics |
treatment: non-infected
cell type: primary macrophages
|
| Treatment protocol |
The primary swine porcine macrophages cultured in six well plates containing 1x10^7 cells per well were infected with recombinant vesicular stomatitis viruses (VSV) at MOI of 10 TCID50 or mock-infected. After infection, the cells were incubated for five hours at 37 °C under 5% of CO2.
|
| Growth protocol |
Cultures of primary swine macrophages were prepared from defibrinated blood as previously described (Borca, M.V., Berggren, K.A., Ramirez-Medina, E., Vuono, E.A., Gladue, D.P., 2018. CRISPR/Cas Gene Editing of a Large DNA Virus: African Swine Fever Virus. Bio-protocol 8, e2978).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from each cell culture well using the RNeasy mini kit (Qiagen) following the manufacturer instructions. RNA quality was assessed using the Agilent 2100 bioanalyzer (Santa Clara, CA, USA) using an RNA nanochip. Quantification of RNA was conducted on a Nanodrop 1000 (Thermo Scientific).
|
| Label |
Cy5
|
| Label protocol |
Low input RNA two color labeling kit (Agilent Technologies) was used to lable cRNA according to the manufacturer's protocol. Total RNA isolated from mock-infection was labeled with Cy5 as universal control. All samples including mock-infection were individually labeled with Cy3.
|
| |
|
| Channel 2 |
| Source name |
VSV-infected (NJ95COB)
|
| Organism |
Sus scrofa |
| Characteristics |
treatment: VSV-infected (NJ95COB)
cell type: primary macrophages
|
| Treatment protocol |
The primary swine porcine macrophages cultured in six well plates containing 1x10^7 cells per well were infected with recombinant vesicular stomatitis viruses (VSV) at MOI of 10 TCID50 or mock-infected. After infection, the cells were incubated for five hours at 37 °C under 5% of CO2.
|
| Growth protocol |
Cultures of primary swine macrophages were prepared from defibrinated blood as previously described (Borca, M.V., Berggren, K.A., Ramirez-Medina, E., Vuono, E.A., Gladue, D.P., 2018. CRISPR/Cas Gene Editing of a Large DNA Virus: African Swine Fever Virus. Bio-protocol 8, e2978).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from each cell culture well using the RNeasy mini kit (Qiagen) following the manufacturer instructions. RNA quality was assessed using the Agilent 2100 bioanalyzer (Santa Clara, CA, USA) using an RNA nanochip. Quantification of RNA was conducted on a Nanodrop 1000 (Thermo Scientific).
|
| Label |
Cy3
|
| Label protocol |
Low input RNA two color labeling kit (Agilent Technologies) was used to lable cRNA according to the manufacturer's protocol. Total RNA isolated from mock-infection was labeled with Cy5 as universal control. All samples including mock-infection were individually labeled with Cy3.
|
| |
|
| |
| Hybridization protocol |
One Cy3 labled sample was cohybrided with the Cy5 labled control in one hybridization chamber (Agilent) containing an Agilent microarray (Array design #:Agilent-069270; 4 X 44k per slide). The chambers were incubated in a incubator at 65 C in a rotating cartridge overnight. After incubation, the arrays were washed with Agilent washing buffers according to amnufacturer's protocol.
|
| Scan protocol |
After hybridization and washing, the arrays were scanned at 5 micrometer per pixel with a Cy3 to Cy5 ratio of approximately 1 using a GenPix 4000B scanner (Molecular Devices) and the images were saved as tiff files.
|
| Description |
pig DNA expression microarray
|
| Data processing |
The expression data were extracted from the tiff files using Agilent Genpix 7.0 software and the raw data were saved as gpr files for statisitcal analysis using R with librarys(limma, MASS, statmod, splines). LOESS method was applied to normalize interarray variation with a background subtraction of 40.
The gpr files were named based on the Agilent array serial number followed by the array number of the slide such as 256927010021-1. The normalized Cy3 results of each sample were used as gene expression levels (photon per pixel) (provided in the Matrix). Gene expression levels of Sample 256927010021-1 were collected from pig #1 macrophages infected with VSV NJ0612NME6. Averaged Expression is the mean expression of each probe. Fold Change is the difference in expression level (fold) between VSV infections. adj.P.Val is false discover rate. WT, M51R and Mock denote VSV NJ0612NME6, NJ0612NME6-M51R and mock infection, respectively.
|
| |
|
| Submission date |
Feb 22, 2023 |
| Last update date |
Apr 02, 2023 |
| Contact name |
James Jiangtao Zhu |
| E-mail(s) |
james.zhu@usda.gov
|
| Phone |
631-323-3186
|
| Organization name |
USDA-ARS
|
| Department |
Plum Island Animal Disease Center
|
| Lab |
Foreign Animal Disease Research Unit
|
| Street address |
40550 Route 25
|
| City |
Orient Point |
| State/province |
NY |
| ZIP/Postal code |
11957 |
| Country |
USA |
| |
|
| Platform ID |
GPL33156 |
| Series (1) |
| GSE225798 |
Understanding the molecular basis leading the virulence of vesicular stomatitis virus in pigs |
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