RNA sequencing and data analysis
Total RNA was also extracted from nasal mucosa tissues using a Nucleo
spin RNA kit (MACHREY-NAGEL, Dueren, Germany). The quantity and quality
of total RNA were evaluated using an Agilent 2100 Bioanalyzer (Agilent,
Santa Clara, CA, USA) and a RNA quality indicator (RIN). Library
preparation was conducted with an Illumina TruSeq Stranded mRNA sample
preparation kit (Illumina, San Diego, CA, USA) according to the
manufacturer′s instructions. Indexing adapters were ligated to the ends
of the cDNA fragments using ligation mix reagent at 30°C for 10 min.
After twice washing with sample purification beads, PCR was performed to
enrich those cDNA fragments that have adapter molecules on both ends.
The quality and band size of the libraries were assessed using an
Agilent 2100 Bioanalyzer. Libraries were quantified by qPCR using the
CFX96 Real Time System (Biorad, Hercules, CA, USA). Sequencing of the
prepared library was conducted on the Hiseq2500 platform (Illumina) with
100 bp paired-end reads.
Reads were trimmed to remove adapters and low-quality reads (per-base
quality < 20) and thereby improve paired-end mapping.
High-quality sequence reads were mapped to the Human genome (hg19) using
STAR,11 and gene expression levels were quantified
with the RSEM (v. 2.12.0).12 Differentially expressed
genes (DEGs) between the 2 groups were evaluated by the edgeR package
(v. 3.0.8),13 which is based on negative binomial
models for RNA-seq count data. DEGs were screened with a cutoff
threshold of log (FC) ≥
|1| and P value < 0.05.