Smart cdna synthesis kit

Total RNAs were extracted from the green and white leaves of R. japonica using the RNeasy Mini kit (Qiagen, Hilden Germany). The quality and concentration of extracted RNAs were determined on a UV spectrophotometer (Nanodrop ND-1000). Gene-specific primers were used for first-strand cDNA synthesis using sequence information for each representative plastid gene (Table S4). The reads from whole-genome sequencing obtained for R. japonica were mapped to the Arabidopsis (Arabidopsis thaliana) ACTIN1 sequence (GenBank accession number: NC_003071) to design gene-specific primers for RjACTIN1 (Table S4). First-strand cDNAs were synthesized using a SMART cDNA Synthesis kit (Takara Bio, Kusatsu, Japan) according to the manufacturer’s protocol.

The qRT-PCR was performed with a 7500 Real Time PCR System (Applied Biosystems) and a SYBR Premix Ex Taq^TM Kit (TaKaRa). Primer pairs were designed against the sequences of selected genes via Primer Express (version 3.0, Applied Biosystems) to amplify target fragments between 150 and 200 bp, and the primers employed for qRT-PCR were listed in Supplementary Table S9. The single-stranded cDNAs were synthesized from 2 μg of the same total RNA applied to microarray hybridization using the SMART cDNA Synthesis Kit (Clontech) and diluted 50-fold with RNase-free water. PCR reactions were conducted in 25-μl volumes containing 1 μl of diluted single-stranded cDNA, 1 × SYBR^® Premix Ex Taq^TM SYBR Green I Master Mix, and 0.5 μM of each primer. Six replicates were carried out in parallel for each gene, and cyclophilin (GenBank: AJ776859) was used as internal control.

The total RNA was isolated using TRIpure reagent (Aidlab, Beijing, China). The obtained organs and hemocytes (10 mg) were homogenized in 1 mL of Trizol reagent for RNA extraction. Based on the previous study in our lab, the cDNA was synthesized using 5 μg total RNA with the SMART cDNA synthesis kit (Clontech) following manufacterer's instuctions. The obtained cDNA was used to detect the expression level of different genes using specific primers (Table 1).

Total RNA was isolated using RNeasy® Plant Mini Kit (Qiagen, Hilden, Germany) following manufacturer's instructions. RNA integrity was visually assessed on a 1% (w/v) agarose gel stained with SYBR® Safe DNA gel stain (Life Technologies, Carlsbad, USA), before proceeding with cDNA synthesis. A total of 250 ng of total RNA from each RNA extraction was combined to provide a total of 1 µg for use in cDNA synthesis using the SMART™ cDNA Synthesis kit (Clontech Laboratories, Mountain View, USA), according to the manufacturer's instructions. Messenger RNA (mRNA) was reverse-transcribed using SMARTScribe™ Reverse Transcriptase and SMART IV™ oligonucleotides. A modified poly-T primer sequence (5′-AAGCAGTGGTATCAACGCAGAGTCGCAGTCGGTACTTTTTTCTTTTTTV-3′) was used instead of the CDS-II primer to reduce sequencing problems associated with the poly-A tails [36] (link).

Reference sequences of CCR1 genes from Sorghum bicolor, Z. mays, P. virgatum and L. perenne were aligned and the consensus sequence was used to design primers to conserved regions. Sequences of all primers are provided as supporting information (Online Resource 1). Total RNA was extracted from 100 mg of ground tissue (stems, roots, leaf blades and inflorescences) from the final reproductive stage of P. dilatatum cv. Primo plants using the RNeasy^® Plant Mini Kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. A cDNA library was prepared from pooled RNA using the SMART™ cDNA Synthesis kit (Clontech Laboratories, Mountain View, CA, USA). First strand cDNA was reverse transcribed using SMARTScribe™ Reverse Transcriptase and SMART IV™ oligonucleotides (Clontech Laboratories) and putative CCR genes were amplified from this template. Further transcripts were identified using the SMARTer™ RACE cDNA Amplification Kit (Clontech Laboratories). After re-amplification with proofreading polymerases, full-length coding sequences were cloned and sequenced using ABI Sanger Sequencing and Big Dye Terminator v3.1 (Life Technologies Corporation, Carlsbad, USA).

Tissues were ground under liquid nitrogen before extraction of total RNA using the Spectrum Plant Total RNA Kit with on-column DNase-treatment (Sigma, Italy) according to the manufacturer’s instructions. RNA concentration and integrity were assessed with a NanoDrop N-1000 spectrophotometer (ThermoFisher Scientific) and standard formaldehyde agarose gel electrophoresis. First-strand cDNA synthesis was performed using the SMART cDNA Synthesis Kit (Clontech Laboratories) and SuperScript III Reverse Transcriptase (Life Technologies).
Double-stranded cDNA was amplified by long-distance PCR using the Advantage^® 2 PCR Enzyme System (Clontech Laboratories). Amplification was performed in a thermal cycler (Applied Biosystems) with the following PCR parameters: 95°C for 1min; 16 cycles of 95°C for 15s and 65°C for 30s; 68°C for 6min; and 4°C for 45min. The double-stranded cDNA was resolved in a 1.1% agarose gel containing ethidium bromide and the cDNA synthesis verified by visualization under UV light.
Samples were sequenced using the 454 GS-FLX instrument according to the manufacturer’s instructions (Roche). Transcriptome data have been published in the GenBank database [PRJNA325155].