概要: 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.
项目整体设计: 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.
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)
处理方案:
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.
提取方案:
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
建库方案:
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.
测序信息
分子类型:
rRNA- RNA
库的片段类型:
PAIRED
库的链类型:
-
测序平台:
ILLUMINA
测序仪型号:
Illumina HiSeq 2000
链特异性:
Unspecific
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文章
Phenotypic responses of differentiated asthmatic human airway epithelial cultures to rhinovirus.