biostudies-arrayexpressFlorian PfaffBos taurusDESeq2TrimGalore (v0.6.6); Salmon (v1.9.0)https://www.ebi.ac.uk/biostudies/studies/E-MTAB-14394In our study, we examined tissue samples from 20 cattle that had been experimentally infected with the FMDV strain O/FRA/1/2001. In 18 of these animals, the infection persisted for more than 28 days. At necropsy, epithelial tissue was collected from the dorsal nasopharynx and dorsal soft palate, the primary sites of persistent infection. Five biological replicates were taken from each location in each animal and analyzed using FMDV-specific RT-qPCR. A subset of these samples was then selected for transcriptome sequencing. Samples were disintegrated using a CP02 cryoPREP (Covaris) and mixed with AL buffer (Qiagen) and TRIzol LS (Invitrogen). After biocontainment removal, trichloromethane was added, and the mixture was centrifuged. About 400 µl of the RNA-containing upper aqueous phase was extracted using the Agencourt RNAdvance Tissue Kit (Beckman Coulter) with a KingFisher Flex processor (Thermo Fisher Scientific). RNA quantity and quality were measured with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). For transcriptomic analysis, 52 samples were selected, including 25 DNP and 23 DSP samples from 14 persistently infected animals and two that cleared the infection before 21 dpc. mRNA was isolated using the Dynabeads mRNA DIRECT Micro Purification Kit (Invitrogen), and libraries were prepared with the Colibri Stranded RNA Library Prep Kit (Invitrogen). ERCC internal control was added before mRNA extraction. The isolated mRNA was fragmented to ~150 nucleotides, adapters were ligated, and the RNA was transcribed into cDNA, which was amplified and indexed for Illumina libraries. The libraries' length and quality were assessed with an Agilent 4150 TapeStation and quantified using Qubit 2.0. Sequencing was performed on a NovaSeq machine in 100 bp single-end mode.biostudies-arrayexpressNucleic Acid Extraction - 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)Sample Collection - 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.Library Construction - 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.Sequencing - For sequencing, a NovaSeq machine (Illumina) running in 100 bp single-end mode was used.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Transcript abundances were analyzed in R (v4.2.1) and imported into DESeq2 (v1.40.2) using the tximport package (v1.28.0). Only transcripts with a relative abundance of at least 10 TPM in at least 2 samples were retained. Prior to principal component analysis (PCA), data were transformed with the DESeq2 “rlog” function. Differentially expressed genes (DEGs) were identified using the DESeq function with various designs to capture contrasts. Log2 fold changes (log2FC) were adjusted with the “lfcShrink” function. The final dataset was filtered with p-value <0.05 and log2FC >|1|.Sequence Alignment - Raw reads were trimmed for quality and adapter contamination using TrimGalore (v0.6.6) and cutadapt (v1.18). A reference genome and transcriptome for cattle (NCBI ARS-UCD1.2; GCA_002263795.2), along with ERCC controls and the FMDV O/FRA/1/2001 sequence, were combined into a single file. A decoy-aware index was created with Salmon (v1.9.0) using the “index” function and used to quantify transcript abundances with the “quant” function, applying corrections for sequence-specific bias and 10 bootstraps.UnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000RNA-seq of coding RNABos taurusFlorian PfafffalseRNA-Seq of persistent foot-and-mouth disease virus infection in the bovine nasopharynxIn our study, we examined tissue samples from 20 cattle that had been experimentally infected with the FMDV strain O/FRA/1/2001. In 18 of these animals, the infection persisted for more than 28 days. At necropsy, epithelial tissue was collected from the dorsal nasopharynx and dorsal soft palate, the primary sites of persistent infection. Five biological replicates were taken from each location in each animal and analyzed using FMDV-specific RT-qPCR. A subset of these samples was then selected for transcriptome sequencing. Samples were disintegrated using a CP02 cryoPREP (Covaris) and mixed with AL buffer (Qiagen) and TRIzol LS (Invitrogen). After biocontainment removal, trichloromethane was added, and the mixture was centrifuged. About 400 µl of the RNA-containing upper aqueous phase was extracted using the Agencourt RNAdvance Tissue Kit (Beckman Coulter) with a KingFisher Flex processor (Thermo Fisher Scientific). RNA quantity and quality were measured with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). For transcriptomic analysis, 52 samples were selected, including 25 DNP and 23 DSP samples from 14 persistently infected animals and two that cleared the infection before 21 dpc. mRNA was isolated using the Dynabeads mRNA DIRECT Micro Purification Kit (Invitrogen), and libraries were prepared with the Colibri Stranded RNA Library Prep Kit (Invitrogen). ERCC internal control was added before mRNA extraction. The isolated mRNA was fragmented to ~150 nucleotides, adapters were ligated, and the RNA was transcribed into cDNA, which was amplified and indexed for Illumina libraries. The libraries' length and quality were assessed with an Agilent 4150 TapeStation and quantified using Qubit 2.0. Sequencing was performed on a NovaSeq machine in 100 bp single-end mode.2025-07-01T00:00:00Z2024-08-29T12:54:21.764Z2024-08-29T12:54:21.764ZE-MTAB-14394ERP163680EFO_0002944EFO_0004170EFO_0004917EFO_0005518EFO_0003816EFO_0003738EFO_0004184