Applied biosystems veriti dx 96 well fast thermal cycler

Total RNA of PDCoV-infected IPEC-J2 cells at 0, 12, and 24 hpi was extracted using a TRIzol reagent kit (Invitrogen, USA) according to the manufacturer’s instructions. RNA integrity (RIN) was measured using an Agilent 2100 Bioanalyzer (Agilent Technologies, Germany), while the purity and concentration of total RNA were measured using a NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific, USA). PDCoV cDNA was obtained using the PrimeScript™ RT reagent Kit (Perfect Real Time) (Takara Biomedical Technology, China) on an Applied Biosystems™ Veriti™ Dx 96-well Fast Thermal Cycler (Thermo Fisher Scientific, USA). Determination of the copy number of the PDCoV M gene was performed using the GoTaq® Probe qPCR Master Mix reagent (Promega, USA) according to the detection method established in our previous study [42 ]. The reactions were performed on an ABI7500 StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, USA). Sequences of forward primer, reverse primer and probe are listed in Additional file 10: Table S10.

Based on transcriptome data analysis and previous studies (Schneweis, Whitfield & Rotenberg, 2017 (link)), we focused on UBR7 (XM_026422690.1). First, the primer sequences were designed using Primer Premier 5.0 software to verify the full-length UBR7 gene (Table 1). Then, according to the instructions of a 2 × TransTaq^® High Fidelity PCR SuperMix I (-dye) kit (TransGen, Beijing, China), PCR amplification was conducted in an Applied Biosystems Veriti™ Dx 96-Well Fast Thermal Cycler (Thermo Fisher, Waltham, MA, USA). The purified PCR products were then ligated into the pClone007 Vector (Tsingke, Beijing, China) and were transformed into Trans5α Chemically Competent Cells (TransGen, Beijing, China). Positive clones were selected by ampicillin resistance and then sequenced by Tsingke Biotechnology, Beijing, China.