Primescript 2 cdna synthesis kit

Total RNA was extracted using TRIzol reagent (Takara Biotechnology, China) and reverse-transcribed to cDNA using a Prime Script II cDNA Synthesis Kit (Takara Biotechnology) according to the manufacturer’s instructions. Real-time quantitative PCR was performed with SYBR-Green master mix (Applied Biosystems, Foster City, CA, USA) in 96-well optical plates using a QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific). GAPDH was used as the reference gene for determination of relative gene expressions. Results are representative of at least three independent experiments.

Blades of the parental sporophytes were collected on the four observation days for RNA extraction. Total RNA was extracted following the method of Yao et al.^{39 (link)}. The extracted RNA was treated with RNase-free DNase I (TaKaRa, Dalian, China) to remove residual genomic DNA. First-strand cDNA was prepared with the PrimeScript II cDNA synthesis kit (Takara, Tokyo, Japan) according to the manufacturer’s instructions and then stored at −80 °C.
Transcriptional analysis of the four Tic20 genes from the parental sporophytes was conducted with real-time quantitative PCR (RT-qPCR). Primer pairs specific to the UTRs (untranslated region) of each of the four Tic20 genes, were used to amplify one 150 -bp amplicon per gene (Table S1). The β-actin primers qActin-F (5′-GACGGGTAAGGAAGAACGG-3′) and qActin-R (5′-GGGACAACCAAAACAAgggCAggAT-3′) were designed as internal controls.
RT-qPCR was performed with the SYBR Premix Ex Taq II kit (Takara, Tokyo, Japan) on the TP800 Thermal Cycler Dice (Takara, Tokyo, Japan). Thermal cycling protocol and data analysis were as previously reported^{40 (link)}.

Total RNA was isolated from 10 weeks old C57BL/6 mice brains using the RNAiso Plus reagents (Takara Bio). First strand cDNA was synthesized using the PrimeScript II cDNA Synthesis Kit (Takara Bio) following the manufacturer’s protocol. The cDNAs encoding the Senp5 open reading frame (ORF) were amplified by Q5 High-Fidelity DNA Polymerase (NEB Japan) with primer sets based on the predicted sequences of the mouse Senp5 isoforms (GenBank accession numbers: Senp5 short isoform 1 [Senp5S], XR_003951796; Senp5 short isoform 2 [Senp5S2], NM_001357088 and XR_004939194). The primers were as follows: forward primer (common to both isoforms), 5′-TCTCGAGCCAGTTCTCATTATGCATCAG-3′; reverse primer for short form 1, 5′-ATGGTACCTTCAGTACAAAGGGAGACAG-3′. PCR products were sequenced and cloned into the XhoI/KpnI sites of the pEGFP-C2 expression vector (Takata Bio). Isolated cDNA clones were designated as Senp5S and Senp5S2. The ORF nucleotide sequences were deposited with the GSDB, DDBJ, EMBL, and NCBI under accession numbers LC619058 (Senp5S) and LC619059 (Senp5S2).

Trizol reagent (Invitrogen, U.S.A.) was used to extract the RNA of tissues and plasma total RNA was isolated using blood total RNA isolation kit (RP4001, BioTeke, Beijing, China) from 300 μl plasma according to the manufacturer’s instructions. The concentration of RNA was measured by Nanodrop 2000 spectrophotometer (Thermo, CA, U.S.A.). The reverse-transcription reaction was performed using PrimeScript II cDNA synthesis kit with gDNA Eraser (Takara, Dalian, China). The reaction conditions for cDNA synthesis were as follows: 42°C for 2 min to remove the contaminated DNA, and then 37°C for 15 min, 85°C for 5 s.

Viral RNAs from cell culture supernatant were extracted using the QIAamp RNA Viral Kit (Qiagen, Heiden, Germany). The extracted RNA was then reverse transcribed to cDNA using the PrimeScript II cDNA synthesis Kit (Takara, Japan). Specific primers were used to amplify the cDNA (Supplementary Table S1) and to subsequently detect SARS-CoV-2, panH1N1, and H7N9. Real-time qPCR experiments were performed using a LightCycler^® 96 system (Roche, Nutley, NJ, USA). The amplification conditions were incubation at 50 °C for 10 min, Taq activation for 5 min at 95 °C, followed by 38 cycles of amplification comprising denaturation for 10 s at 95 °C, annealing, and primer extension for 40 s at 55 °C.

Total RNA was extracted from hypopharyngeal cancer (HPCM2, HPCM7) cells using the RNeasy Mini Kit (Qiagen, Valencia, CA) or Sepasol-RNA I Super G (Nakalai Tesque, Japan), and reverse transcribed using a PrimeScript II cDNA Synthesis Kit (Takara Bio, Japan). Real-time PCR was performed using the Brilliant III Ultra- Fast SYBR Green QPCR Master Mix (Agilent Technologies). ß-actin was used as an endogenous reference gene. The primer sequences used for real-time PCR are listed in Supplementary Table S2.