Gene Expression
Nebulas
Gene Expression NebulasSummary: We report the application of RNA sequencing technology for high-throughput profiling of gene expression responses to human rhinovirus infection at 24 hours in air-liquid interface human airway epithelial cell cultures derived from 6 asthmatic and 6 non-asthmatic donors. RNA-seq analysis identified sets of genes associated with asthma specific viral responses. These genes are related to inflammatory pathways, epithelial remodeling and cilium assembly and function, including those described previously (e.g. CCL5, CXCL10 and CX3CL1), and novel ones that were identified for the first time in this study (e.g. CCRL1, CDHR3). We concluded that air liquid interface cultured human airway epithelial cells challenged with live HRV are a useful in vitro model for the study of rhinovirus induced asthma exacerbation, given that our findings are consistent with clinical data sets. Furthermore, our data suggest that abnormal airway epithelial structure and inflammatory signaling are important contributors to viral induced asthma exacerbation.
Overall Design: Differentiated air-liquid interface cultured human airway epithelial cell mRNA profiles from 6 asthmatic and 6 non-asthmatic donors after 24 hour treatment with either HRV or vehicle control were generated by deep sequencing, using Illumina HiSeq 2000.
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| Growth Protocol: | ALI cultures (0.6 cm2 insert devices from Millipore cultured in 6 well plates) were changed to hydrocortisone-free media for 24 hours (37ºC, 5% CO2) |
| Treatment Protocol: | The apical surface of ALI cultures was washed twice with room temperature PBS to remove accumulated mucus prior to apical treatment application of HRV16 at MOI=10 or culture media. Treated cultures were shifted to a 34ºC incubator (5% CO2) with gentle shaking on a Bellco orbital shaker until 24 hours. |
| Extract Protocol: | Cells were washed three times with PBS, followed by adding 175 uL RLT buffer to each ALI device ALI cell lysates were transferred to tubes on ice, and were further rinsed and samples were pooled with an additional 175 uL aliquot of RLT (350 uL total/sample). Total RNA was isolated from RLT cell lysates using the QIAGEN RNeasy mini kit, and quantified via Nanodrop (Thermo Scientific, Wilmington, DE) Kit |
| Library Construction Protocol: | RNA libraries were prepared for sequencing using standard Illumina protocols. Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols. |
| Molecule Type: | rRNA- RNA |
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| Library Layout: | PAIRED |
| Library Strand: | - |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 2000 |
| Strand-Specific: | Unspecific |
| Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
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