Hiscriptii1st strand cdna synthesis kit

The RaPure viral RNA/DNA Kit (Magen, Guangzhou, China) was used to extract nucleic acids from clinical samples. The extracted total nucleic acids were used for RNA reverse transcription (RT) using HiScriptⅡ1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) and amplification. The reaction system volume of RT was 20 µL, consisting of 10 µL of 2 × RT Mix, 1 µL of random hexamers, 7 µL of total RNA and 2 µL of HiScript Ⅱ Enzyme Mix. Finally, the cDNA was stored at −20 °C.
The reaction system volume of mPCR for PAIT and PAFR was 20 µL, including 10 µL of 2 × Taq Master Mix (Vazyme, Nanjing, China), 1 µL of template, 1 µL of the forward primer (10 mM), 1 µL of reverse primer (10 mM) and 7 µL of ddH₂O. The single PCR program was as follows: 98 °C for 3 min; 36 cycles of 98 °C for 20 s, 57 °C for 1 min, and 72 °C for 1 min 30 s; and 72 °C for 5 min. The products of PCR were detected with 2% agarose gel electrophoresis. The PCR amplification fragments of FCV (1940 bp), FHV-1 (1014 bp), FeLV (608 bp), C. felis (500 bp), IAV (155 bp), FCoV (726 bp), FeAstV (418 bp), FPV (337 bp) and FeKoV (198 bp) were ligated into the pMD 18-T vector (TaKaRa, Dalian, China). The ligation products were transformed and cultured, and then the positive plasmids were extracted. Additionally, the positive test plasmids were sent to the Beijing Genomics Institute (Guangzhou, China) for sequencing.

RNA of tissues (hippocampus and cortex) was extracted with Trizol (Sangon Biotech, Shanghai, China). Total RNA quantity was measured using a BioPhotometer (Eppendorf, Hamburg, Germany). Reverse transcription from 2 µg of RNA was performed using the Hiscript Ⅱ 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). Real-time PCR was performed using Power Syber Green PCR Master Mix (Vazyme, Nanjing, China) and detected by the LightCycler Instrument (Roche Diagnostics, Basel, Switzerland) with software version 1.5.1.62. Results were normalized to the expression of the housekeeping gene Actin. The cycling parameters were as follows: stage 1, 95 °C for 5 min; stage 2, 40 cycles of 95 °C for 10 s and 60 °C for 30 s; stage 3, 95 °C for 15 s, 60 °C for 60 s and 95 °C for 15 s, which were concluded by the melting curve analysis process, and fold changes of gene expression were calculated using the 2−ΔΔCt method. The sequences used in this analysis were as follows: Tnf: forward 5′-CACGCTCTTCTGTCTACTGAACTTC-3′, reverse 5′-GCAGCCTTGTCCCTTGAAGAGAACC-3′; Il1b: forward 5′-GCAACTGTTCCTGAACTC-3′, reverse 5′-CTCGGAGCCTGTAGTGCA-3′; Actb: forward 5′-CATCCGTAAAGACCTCTATGCC-3′, reverse 5′-GACTCATCGTACTCCTGCTTG-3′.

The candidate DEGs were selected to carry out a tissue-specific expression analysis by qRT-PCR. The gene-specific primers were designed and assessed using the Primer Premier 5 software tool. Total RNA was reverse transcribed by the manufacturer’s recommendation using HiScript^® Ⅱ 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). A total of 2 µL of cDNA products were used as templates in a 20 µL qPCR reaction system. The Quantitative Real-time PCR reactions were performed on the QuantStudio™ 6 Flex Real-Time PCR System (Applied Biosystems, MA, USA) using a TransStart Top Green qPCR SuperMix (TransGen, Beijing, China). Each sample had three technical replicates. SmActin was used as an endogenous control to normalize the expression value. All the primers used are listed in Table S1. The relative quantitation was calculated by the Comparative CT (2^-−ΔΔCT) method.