Cfx384 real time system c1000 touch thermal cycler

The EASYspin Plus Complex Plant RNA Kit (RN53) (Aidlab Biotechnology, Beijing, China) was used for the RNA isolation. The PrimeScript™ RT Reagent Kit with gDNA Eraser (TaKaRa, Shiga, Japan) was used to synthesize the single-stranded cDNA. Quantitative real-time PCR (qRT-PCR) was performed using an CFX384-Real-Time System (C1000 Touch Thermal Cycler, Bio-Rad, Irvine, CA, USA) with the RealUniversal Color PreMix (SYBR Green) (TIANGEN, Beijing, China). The specific primers for qRT-PCR were designed at NCBI (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) (accessed on 20 October 2022). Actin was selected to be the internal control for gene expression analyses [65 (link)]. The primers were listed in Table S1. All experiments were conducted in three biological replicates. The comparative 2^−ΔΔC_T method was used to calculate the relative expression levels of each transcript [66 (link)].

To quantify viral RNA abundance in the supernatant samples, RT-qPCR was performed with RNA standards synthesized using a TranscriptAid T7 High Yield Transcription Kit (Thermo Scientific Cat# K0441) from the DNA templates generated by RT-PCR from the SARS-CoV-2 and 229E viral RNA. For each sample, total RNA was isolated using a Quick-RNA Viral kit (Zymo Research Cat# R1035). RT-qPCR was performed using the GoTaq Probe 1-Step RT-qPCR System (Promega Cat# A6120). RT-qPCR primers and probes are listed in Supplementary Data 2.
To quantify viral RNA abundance in the cell samples, RT-qPCR was performed using the same primers employed for the supernatant viral load. ACTB (beta-actin) mRNA levels served as an internal control. RT-qPCR was performed using the iScript™ cDNA Synthesis Kit and iTaq Universal SYBR Green Supermix (Bio-Rad) and run on a Biorad CFX384 real-time system (C1000 Touch Thermal Cycler), according to the manufacturer’s instructions. The relative viral RNA abundance was normalized to the ACTB internal control. The relative viral RNA abundance of each treatment was normalized to NT samples.

To analyze gene expression in infected and treated macrophages, RTqPCR was performed. RNA was isolated using the RNeasy mini kit (Qiagen) and cDNA was generated according to the manufacturer’s instructions with the GoScript Reverse transcription system (Promega). qPCR was performed with SsoFAST EvaGreen Supermix (BioRad) on the CFX384™ Real-time System C1000 Touch™ Thermal Cycler (BioRad) and analyzed using the CFX Maestro software (BioRad). Expression of the housekeeping gene reep5 was analyzed to enable normalization, and data are represented as a fold-change relative to the UI control. All reagents and primers are listed in Supplementary Table 1.

The Tm was determined using a CFX384™ Real-Time System C1000 Touch™ Thermal Cycler (Bio-Rad, Hercules, CA, USA). Briefly, 18 μL of 0.1 mg/mL sample was mixed with 2 μL of 100× Sypro orange protein gel stain (Invitrogen). The plate was ramped from 30.0 °C to 99.6 °C with 0.6 °C/step, and the temperature was held for 2 s for the subsequent step. The Tm was assigned using the first derivative of the raw data. To compare the stability of antibodies before and after the introduction of FAST-Ig mutations, the first peak of the melting curve (Tm1) was referenced.

Knockdown efficiencies of targeting siRNAs were evaluated by reverse transcription qPCR 3-days after siRNA transfection. For relative quantification of lytic gene expression, cells transfected with siRNAs were untreated or treated with 1 μg/ml DOX plus 1 mM NaB at 3-days post transfection and harvested at multiple time points following lytic induction; 8h for RTA, 24h for mCherry and ORF10 and 48h for K8.1. The latent condition analysis for RTA (Fig 5A) was harvested 3-days post transfection with siRNAs. Total RNA was extracted from cells following the RNAeasy Mini Kit (QIAGEN #74104) protocol and cDNA was synthesized from RNA samples according to the High-capacity RNA-to-cDNA (AppliedBiosystems #4387406) protocol. Quantitative PCR of cDNA samples were carried out using Power Sybr Green Master Mix (ThermoFisher #4367659) on a Bio-Rad CFX384 Real Time System C1000 Touch Thermal Cycler using primers listed in S8 Table. Relative mRNA levels were normalized to tubulin control and calculated using the ΔΔCt method for experimental conditions as compared to control conditions.

Total RNA was extracted from tissue samples using Trizol (Invitrogen 15596026) following the manufacturer’s instructions. 2000ng RNA per sample was used to generate cDNA using Applied Biosystems High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems 4368814) following the manufacturer’s instructions. cDNA was diluted to 1:5 in water before performing qPCR using Intact Genomics ig SYBR Green 2x Master Mix (Intact Genomics 3354). qPCR was carried out with the CFX384 Real-Time System C1000 Touch Thermal Cycler (Bio-Rad Laboratories). Cycling conditions were as follows: Initial denaturation 95°C for 2 minutes, 40 cycles of denaturation at 95°C for 5 seconds followed by annealing/extension at 60°C for 30 seconds. Primers used are listed in Table S3. Relative expression was calculated using the ΔΔCt method, using β-actin as the reference gene.