title: 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)
design: 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)
library_name: O-2-24-T1_p
library_construction_protocol: Crypts were isolated from jejunum of stillborn piglets and put in culture in matrigel (Corning) and Organoid Growth Medium with a ratio 1:1 of Supplement and basal mediums (Stem Cell Technology). After 8 days of culture, 3D organoids were formed. To maintain the 3D organoids culture, the cells were dissociated and diluted in 1:4 to add matrigel and Organoid Growth Medium with a ratio 1:1 of basal and Supplement mediums (Stem Cell Technology). This protocol was maintained during 5, 14 and 25 passages. At passage 25, 3D organoids were dissociated and transfered in matrigel coated plated to spread the cells and produced 2D organoids cultivated in Organoid Growth Medium with a ratio 1:0.8 of basal and Supplement mediums (Stem Cell Technology). 2D organoids (after 3 days of plating) were washed twice with DMEM F12 and were inoculated with TGEV at multiplicity of infection of 0.06 and 1. The cells were washed twice after 1 hour of inoculation and Organoid Growth Medium with a ratio 1:0.8 of basal and Supplement mediums (Stem Cell Technology) was added. The corresponding controls were mock-infected with ST cells supernatant. 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 Illumina Stranded mRNA Kit, according to the supplier's recommendations. Essentially, the mRNA were first enriched by the capture of polyA tails via oligoDT. After enzymatic fragmentation, 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.