RNA-Seq of persistent foot-and-mouth disease virus infection in the bovine nasopharynx
RNA-Seq of persistent foot-and-mouth disease virus infection in the bovine nasopharynx
Nasopharyngeal tissue samples were collected during necropsy. Five replicates from each DNP and DSP epithelium were taken to capture the focal distribution of FMDV infection. To preserve mRNA integrity, samples were frozen in liquid nitrogen and stored at -80°C. Samples were disintegrated using a CP02 cryoPREP (Covaris) and mixed with 250 µl AL buffer (Qiagen) and 750 µl TRIzol LS (Invitrogen). After biocontainment removal, 200 µl trichloromethane was added, and the mixture was centrifuged. The upper aqueous phase, containing RNA, was extracted and processed with the Agencourt RNAdvance Tissue Kit (Beckman Coulter) using a KingFisher Flex (Thermo Fisher Scientific). RNA quantity and quality were assessed with a NanoDrop 1000 (Thermo Fisher Scientific) For transcriptomic analysis, 52 samples were selected for sequencing: 25 DNP and 23 DSP samples from 14 persistently infected animals and 2 that cleared the infection before 21 dpc. mRNA was isolated from total RNA using the Dynabeads mRNA DIRECT Micro Purification Kit (Invitrogen). Library preparation was done with the Colibri Stranded RNA Library Prep Kit for Illumina (Invitrogen), and ERCC internal control (Invitrogen) was used before mRNA extraction as recommended. mRNA was fragmented to ~150 nucleotides using RNase III, then adapter-ligated and transcribed into cDNA with 10× SuperScript IV Enzyme Mix. The cDNA was amplified with index primers across 12 or 13 cycles to generate barcoded Illumina libraries. Library length and quality were checked on an Agilent 4150 TapeStation and quantified with a Qubit 2.0 and Qubit dsDNA HS Assay Kit. Libraries were pooled at an equimolar ratio.