Transcriptomics analysis of different porcine intestinal models (piglet jejunums, jejunum crypts, and organoids) and conventional cell line models upon infection with the transmissible gastroenteritis virus (TGEV)
Transcriptomics analysis of different porcine intestinal models (piglet jejunums, jejunum crypts, and organoids) and conventional cell line models upon infection with the transmissible gastroenteritis virus (TGEV)
Each experimental group of Specific Pathogen-Free (SPF) piglets was housed in a separate room in a ABSL3 facility. After 24hpi and 48hpi, different organs from the piglets, including the jejunum, duodenum, ileum, colon, lung, trachea, bronchi, trachea-bronchial and mesenteric lymph node, were harvested and placed in RNAlatter (ThermoFisher) to preserve RNA quality. Specific Pathogen-Free (SPF) piglets were oro-nasally inoculated with 7 mL of EMEM (2 mL nasal and 5mL in the oral route) for the control piglets or with 10^4.75 TCID50 per piglet, corresponding to 10^8 copies of viral N gene per pig for the infected piglets. The total RNA of jejunum crypts, 3D organoids, 2D organoids, piglet jejunums and ST cells were extracted with Trizol Reagent, according to the supplier's recommendations. After Turbo DNAse treatment and validation of RNA quality, 500ng of RNA were used to build the sequencing librairies. Libraries were generated with the Total RNA Library Prep Set Kit, according to the supplier's recommendations. Essentially, the mRNA were first enriched by the capture of polyA tails via oligoDT. After fragmentation per sonication, the cDNA were synthetized and the protruding ends of the cDNA were converted into blunt ends by exonuclease/polymerase activities. The 3' ends were adenilated and the adaptators were ligated. The libraries were amplified by PCR reactions and sequenced.