Next Generation Sequencing of Lung Primary Fibroblast Responding to Eosinophil-Degranulation Products

Methods: Two human lung primary fibroblast (HLF) lines were activated for 24 h with eosinophil conditioned media obtained from two blood eosinophil donors. Total RNA was extracted from HLF and treated with DNase. RNA quality was analyzed via Agilent 2100 Bioanalyzer platform. The sequencing library from mRNA was prepared using TruSeq Stranded mRNA Library Preparation Kit (Illumina; San Diego, CA, USA) and RNA-seq (1x100 bases; 25 million reads per sample) was carried out using Illumina HiSeq 2500 platform. SeqMan Ngen (v.13) and ArrayStar with Qseq module (v.13) software packages (DNAStar, Madison, WI) were used to map sequence reads to human reference genome (GRCh38), apply statistical analyses and identify global gene expression changes. Results: Compared to control HLF cultures, conditioned medium from IL-3-primed degranulated eosinophils on IgG upregulates 169 genes and downregulates 131 genes in both HLF lines. Conclusions: Our study represents the first detailed analysis of gene expression in HLF activated with degranulated products from eosinophils, generated by RNA-seq technology. Analyses of the genes which expression level is changed by conditioned media from degranulated eosinophils should inform on the function of eosinophils on fibroblasts. Human lung primary fibroblast mRNA profiles after activation with conditioned media from degranulated eosinophils were generated by deep sequencing, using Illumina HiSeq 2500 platform. Two eosinophils donors and two HLF lines were used.