RNA-seq comparison of gene induction in RIG-I stimulated old and young human monocytes

Goal: The goal of this study was to compare gene induction in old and young human monocytes upon stimulation of these cells with a RIG-I specific ligand, based on previously published results which indicated impaired interferon responses in monocytes from older individuals upon influenza virus infeciton. Overall design: Methods: We isolated monocytes from human donor peripheral blood of young (n = 2) or old (n =3) donors. Cells were then transfected with a 14bp hairpin RNA construct bearing a 5'-ppp motif that specifically stimulates RIG-I. After 6 hours, total RNA was collected from stimulated cells and RNA was purified using the RNeasy kit (Qiagen) according to manufacturer's protocols. RNA was isolated from cellular lysates in the same manner as for qPCR experiments. RNA sequencing was performed by the HudsonAlpha Institute for Biotechnology. RNA samples were sequenced using an Illumina HiSeq 2500 machine, with 125 base pair reads. The raw reads of RNA-seq experiments were trimmed off sequencing adaptors and low quality regions by btrim. The trimmed reads were mapped to human genome (GRCh37) by tophat2 . The counts of reads for each gene were based on Ensembl annotation (release 70). After the counts are collected, the differential expression analysis was done by DEseq2, which calculated the adjusted p-values.