GSM5315968: 503A-CD163-eGFP-minus; Sus scrofa; RNA-Seq
On 7 dpi animals from both DMEM and PRRSV treatment groups were euthanized. Immediately post-euthanasia, a single-cell suspension of PAM cell was isolated aseptically and sorted in to infected and bystander group based on EGFP expression. Sorted PAM cells stored at -80 0C in TRIzol (ThermoFisher Scientific). RNA extraction, RNA library preparations, sequencing reactions, and initial bioinformatics analysis were conducted at GENEWIZ, LLC. (South Plainfield, NJ, USA). Total RNA was extracted following the Trizol Reagent User Guide (Thermo Fisher Scientific). RNA was quantified using Qubit Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked with TapeStation (Agilent Technologies, Palo Alto, CA, USA). SMART-Seq v4 Ultra Low Input Kit for Sequencing was used for full-length cDNA synthesis and amplification (Clontech, Mountain View, CA), and Illumina Nextera XT library was used for sequencing library preparation. Briefly, cDNA was fragmented, and adaptor was added using Transposase, followed by limited-cycle PCR to enrich and add index to the cDNA fragments. The final library was assessed with Agilent TapeStation. The sequencing libraries were multiplexed and clustered on one lane of a flowcell. After clustering, the flowcell was loaded on the Illumina HiSeq instrument according to manufacturer's instructions. The samples were sequenced using a 2x150 Paired End (PE) configuration. Image analysis and base calling were conducted by the HiSeq Control Software (HCS). Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mis-match was allowed for index sequence identification.