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Status |
Public on Sep 11, 2013 |
Title |
Let1_miR144_1h.5 |
Sample type |
RNA |
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Source name |
immortalized airway epithelial cells, expressing miR-144, infected with PR8 influenza 1 hour
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Organism |
Mus musculus |
Characteristics |
cell type: immortalized airway epithelial cells transgene: miR-144 time: 1 hour infection: PR8 influenza strain: C57BL/6
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Treatment protocol |
Cells were infected with influenza A (A/Puerto Rico/8/34) (PR8) at MOI=5 in OptiMEM in the absence of trypsin, the inoculum removed by washing, and cells cultured in OptiMEM.
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Growth protocol |
To generate LET1 cells, lung cells were isolated from C57Bl/6 mice using a modified dispase-agarose protocol (Herold et al., 2006), cultured to yield lung epithelial type I cells (LET1) trans-differentiated from type II epithelial cells, then immortalized by transduction with MSCV-SV40 large T antigen and used after a minimal number of passages. LET1 cells were cultured in DMEM containing 10% heat-inactivated FBS, 2 mM L-glutamine, and penicillin-streptomycin (complete DMEM), incubated at 37oC 5% CO2., passaged using a brief trypsinization, and sub-cultured 1:20 every 3 days.
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was isolated from bone-marrow-derived macrophages using TRIzol (Invitrogen) according to the manufacturer's instructions. Prior to labeling, the integrity of samples was checked using an Agilent 2100 Bioanalyzer.
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Label |
Cy3
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Label protocol |
Labeling was performed with the One-Color Microarray-Based Gene Expression Analysis protocol version 6.5 (Agilent). Briefly, double-stranded cDNA was synthesized from 200ng of total RNA using a T7-oligo(dT) primer, followed by in vitro transcription by T7 RNA polymerase with incorporation of Cy3-labeled CTP.
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Hybridization protocol |
600ng of Cy3-labeled cRNA was hybridized in Agilent hybridization chambers for 17 hours at 65 C and 10rpm. The arrays were washed for 1 minute in Agilent GE Wash Buffer 1 and then for another minute in Agilent GE Wash Buffer 2.
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Scan protocol |
Scanning was performed using an Agilent High-Resolution DNA Microarray Scanner, with 3 micron resolution and 20-bit dynamic range. The resulting images were processed with Agilent Feature Extraction version 10.7.3.1 to generate raw probe intensity data.
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Description |
Gene expression in miR-144 expressing cells infected with PR8 influenza for 1 hour
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Data processing |
Arrays were quantile normalized using the normalizeBetweenArrays function from the Bioconductor marray package.
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Submission date |
Sep 10, 2013 |
Last update date |
Sep 11, 2013 |
Contact name |
Aderem Lab |
E-mail(s) |
alan.diercks@seattlechildrens.org
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Organization name |
Seattle Children's Research Institute
|
Lab |
Aderem Lab
|
Street address |
307 Westlake Avenue North, Suite 500
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City |
Seattle |
State/province |
WA |
ZIP/Postal code |
98109 |
Country |
USA |
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Platform ID |
GPL13912 |
Series (1) |
GSE50742 |
Effect of miR-144 and miR-451 expression on lung epithelial cell responses to influenza infection at 1 and 18 hours. |
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