Duck embryo fibroblast cell gene expression during the early stage of egg drop syndrome virus infection
DEFs were obtained from 12-day-old duck embryos according to a previously described method (Huang, et al., 2015).  DEFs were cultured in plates containing Dulbeccos modified Eagles medium (DMEM, Gibco, US) supplemented with 10% fetal bovine serum (FBS, Gibco, US) at 37 C in a 5% CO2 atmosphere. EDSV strain 127 (GenBank: Y09598) was used in this study according to a previously described method (Huang et al., 2015).DEFs were infected with EDSV at a multiplicity of infection (MOI) of 2.Cellular and viral RNA was extracted with TRIzol reagent (Invitrogen, Life Technologies, USA) according to the manufacturer's protocol and subsequently treated with DNase I (Invitrogen, Life Technologies, USA). RNA purity, concentration and integrity were assessed using a Nanodrop NanoPhotometer spectrophotometer (NanoDrop products IMPLEN, CA, USA), a Qubit 2.0 fluorometer (Invitrogen, Life Technologies, USA) and an Agilent 2100 bioanalyzer (Agilent, USA), respectively. A cDNA library for each sample was constructed with an RNA Library Prep Kit (NEB, USA) according to the manufacturer's instructions. Next, 3 ?g of RNA was used to enrich poly (A) mRNA, which was fragmented into short pieces by oligo (dT) magnetic beads (Invitrogen, USA). Cleaved short RNA fragments were used for first-strand cDNA synthesis with reverse transcriptase and a random hexamerCprimer. Second-strand cDNA fragments were then synthesized using DNA polymerase I, RNase H, and dNTPs. The cDNA fragments were ligated to sequencing adapters and amplified by PCR to obtain the final paired-end (PE) library. Library sequencing was performed on a BGIseq-500 platform at the Beijing Genomic Institution.