Universal riboclone cdna synthesis system

Freshly isolated brains were microdissected on ice to isolate the hypothalamus. Total RNA was extracted using the TRIzol method, and first‐strand cDNA was synthesized using a Universal RiboClone cDNA Synthesis System (Promega; Catalog # C4360). mRNA expression was assessed with NovaTM SYBR Green PCR Master Green mix (Qiagen; Catalog # 204143), and analysis was performed using the 2‐∧∧CT method. The results for all qPCR samples were normalized to actin.

Because the short style is controlled by the S allele (see the Introduction), thrum plants of ‘HTC’ were used for cloning. Genomic DNA was isolated from young leaves with a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Total RNA was isolated from buds, mRNA was isolated from total RNA with a Micro-Fast Track Kit (Invitrogen, Carlsbad, CA, USA), and cDNA was synthesised with the Universal Ribo Clone cDNA synthesis system (Promega, Madison, WI, USA). Based on the N-terminal and internal partial amino acid sequences, degenerate primers were designed and used to obtain partial cDNA and genomic DNA fragments. Thermocycling conditions were as follows: initial denaturation at 94 °C for 2 min; 35 cycles of 94 °C for 30 s, 42 °C for 30 s, 72 °C for 1 min; and final extension at 72 °C for 5 min. Amplified DNA fragments were cloned with the TA Cloning Kit (Invitrogen) and sequenced on an ABI3100 sequencer (Applied Biosystems, Waltham, MA, USA). We performed 3ʹ RACE with the 3ʹ-Full RACE Core Set (Takara, Otsu, Japan) to determine the sequence of the 3ʹ region of the gene, and then conducted genome walking with the GeneRacer Kit (Invitrogen) to determine the sequence of the 5ʹ region of the gene. Primers are listed in Supplementary Table S2.

Aliquots of 5 μl of the purified DNA solution obtained with monoclonal anti-SIRT1 antibody were treated with T4 DNA polymerase and dNTPs (Roche Applied Science, Mannheim, Germany) for 15 min at 16°C and subsequently heat inactivated at 75°C for 10 min. According to the manufacturer’s instructions, EcoRI adaptor oligonucleotides were ligated to the DNA pieces and excess adaptors subsequently removed with Sephacryl S-400 spin columns (Universal Riboclone cDNA Synthesis System, Promega, Madison, WI, USA). Purified DNA was amplified by standard PCR, using primer matching the EcoRI adaptor sequence (forward and reverse primer: AATTCCGTTGCTGTCG). Amplified DNA was briefly separated on 1.5% agarose gels from which a fragment range between approximately 200 bp–500bp was extracted (Qiagen, Valencia, CA, USA). Purified DNA was ligated into the pGEM-T Easy vector system and transformed into E. coli JM109 (Promega, Madison, WI, USA).

Viral RNA samples from purified virions served as a template for cDNA synthesis using random hexamer and oligo dT₁₇VN primers with a Maxima reverse transcriptase (Fermentas). The cDNA served as a template for the second strand synthesis using the Universal RiboClone cDNA Synthesis System (Promega, Madison, WI, USA), according to the manufacturer’s instructions. The ds-cDNA was purified using the Zymoclean kit (Zymo Research, Irvine, CA, USA), and the obtained double-stranded fragments were cloned as a library into a commercial pUC19/SmaI (Fermentas). The library was then transformed into DH5α competent cells, and insert-positive colonies were cultured in LB media, including antibiotics. The plasmids were extracted using a plasmid extraction kit (Bioneer, Daejeon, Republic of Korea) and sequenced by Sanger sequencing (HyLabs, Rehovot, Israel).
Based on the obtained sequences, the primer pairs were designed (Table 1) and used for RT-PCR to obtain the unidentified genome segments. The 5′ end of the genome was identified using the RACE strategy on cDNA derived from viral RNA extractions, while the 3’ end was sequenced using the oligo dT₁₇VN primer.

RNA sample was reversely transcribed to cDNA according to instruction of Universal RiboClone^® cDNA Synthesis System (Promega, Beijing, China). qPCR was conducted using TB Green Premix Ex Taq II (TliRNaseH Plus) (RR420Q TaKaRa Biotechnology, Beijing, China) on an ABI7500 system (Thermo Fisher, Singapore, Asia). The reaction procedure was completed with the following protocol: 95 °C 3 min, 45 cycles of 5 s at 95 °C, 30 s at 60 °C and 71 cycles of 15 s at 60 °C. All samples were tested with three technical replicates and three independent biological replicates. The relative expression level was calculated while using the 2^−∆∆CT method (Livak & Schmittgen, 2001 (link)). According to a previous study in P. cornutum, the beta-actin gene was regarded as a reference gene (Wang, Wang & Yang, 2017 (link)).