Rnase free dnase

The total RNA for each sample was extracted with the RNAprep Pure Plant Kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s protocol. RNA was further purified using RNase-free DNase (TIANGEN) according to the manufacturer’s guidelines. The quality, quantity and integrity of the RNA samples were assessed as described previously^{3 (link)}. cDNA was synthesized from 300 ng RNA using the PrimeScript II First Strand cDNA Synthesis Kit MIX (Bioteke, Beijing, China) with oligo (dT) primers in a final volume of 20 μL according to the manufacturer’s instructions.

Genome DNA and floral transcriptomic RNA were isolated from Hybrid Tea Rose ‘Samantha’ leaves for paired-end sequencing on the Illumina 150 × 2 platform. ‘Samantha’ produces scented, medium-sized red flowers and dark green, leathery foliage, for which it is cultured worldwide [15 (link)]. Genomic DNA was isolated from young leaves of rose plants as described by Aldrich and Cullis (1993) [16 (link)], but with 1% (w/v) polyvinylpyrrolidone-10 added to the DNA extraction buffer. Total RNA was extracted from rose floral organs using a TRIzol RNA Purification kit (TaKaRa, China) following the manufacturer’s instructions. The RNA was treated with RNase-free DNase (Tiangen, China) to remove residual genomic DNA. First-strand cDNA synthesis was conducted using 20-μL reaction mixtures containing 1 μg of total RNA and oligo(dT) primers (TaKaRa, China).

Roots of Col-0 were harvested to extract total RNA for real-time PCR. Total RNA was extracted using RNAprep pure plant kit (Tiangen, Beijing) and treated with RNase free DNase (Tiangen). The total RNA was reverse-transcribed into first-strand cDNA using PrimeScript™ Reverse Transcriptase (Takara, Japan) and Oligo (dT)₁₅ primer (Takara) following the manufacturer's instructions. The samples were amplified using SYBR Green I (SYBR® Premix Ex Taq™ Kit, Takara). The housekeeping gene EF1A was used as an internal control. The thermal cycle used was as follows: 95°C for 10 s, and 40 cycles of 95°C for 5 s and 59°C for 25 s. This was followed by 80 cycles of 10 s during the time elapsed during 55–95°C. The PCR amplifications for each gene were performed in triplicate. The results were analyzed by Rotor-Gene Real-Time Analysis Software 6.1 (Build 81). All the primers used in this study were shown in Table S1.

Total RNA for each sample was isolated using the RNAprep pure Plant Kit (Tiangen, Beijing, China) and treated with RNase-free DNase (Tiangen) in accordance with the manufacturer’s protocol. The quality, quantity, and integrity of the total RNA extracted were assessed as previously described [49 (link)]. Total RNA was reversed transcribed into cDNA using the Superscript First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). The qRT-PCR using the SYBR Green Master Mixture (CWBIO, Beijing, China) on an Applied Biosystems 7500 Fast Real-Time PCR System (Life Technologies, Foster City, CA, USA) were performed as previously described [53 (link)]. The PCR amplification system and program were as described previously [52 (link)]. Primers used in this study are listed in Additional file 16: Table S8. All qRT-PCR assays were repeated three times as independent biological replicates. Relative gene expression levels were calculated using the 2^–∆Ct method [54 (link)].

GEFs were synchronized for 2 h with 100 nM dexamethasone (DXM, Sigma-Aldrich, St. Louis, MO, USA) in serum-free DMEM/F-12 medium containing 1 × AA. Cell samples were harvested at the indicated time points (after DXM synchronization for 24, 28, 32, 36, 40, or 44 h). Total RNA was extracted and purified from GEFs and goat liver and kidney tissues using TRIzol reagent (TaKaRa, Dalian, China) and then treated with RNase-free DNase (TianGen, Beijing, China). cDNA was generated using a PrimeScript RT Reagent Kit (TaKaRa). The primer sets used for the qPCR are listed in Table 1. The qPCR reactions were carried out in a 20 μL reaction solution comprising 10 ng cDNA with specific primers (200 nM) using the Thunderbird SYBR qPCR Mix (Toyobo, Osaka, Japan) and a CFX96™ qRT-PCR system (Bio-Rad, Hercules, CA, USA), as described previously (34 (link), 35 (link)). All reactions were performed in triplicate. The relative expression levels of each sample were normalized to the average level of the constitutively expressed housekeeping gene, RPLP0 (also known as 36B4).

All strains were cultured at mid-exponential, harvested, washed with PBS. Total RNA was isolated from each sample using the RNAprep Pure Cell/Bacteria Kit, treated with RNase-free DNase (Tiangen) and measured the purity and concentration by gel electrophoresis and spectrophotometer (NanoDrop, Thermo Scientific). 1.5 μg of total RNA was used to synthesis the first-strand cDNA by reverse transcriptase (TransGen Biotech). Quantitative real-time PCR (qRT-PCR) was performed in CFX96 Real-Time PCR Detection System (Bio-Rad) with TransStart Green qPCR SuperMix (TransGen Biotech). The qRT-PCR primers were listed in Supplementary Table S2 and qRT-PCR parameter was set as follows: 95°C for 30 s followed by 40 cycles of 94°C for 15 s, 52°C for 30 s. The relative abundance of 16S rRNA was used as the internal standard for standardization of results.

Total RNA was extracted as indicated above. Each RNA sample was treated with RNase-free DNase (Tiangen, Beijing, China) following the manufacturer’s protocol in an effort to remove any residual genomic DNA (gDNA). DNase-treated RNA (2 mg) was subjected to reverse transcriptase reactions using M-MLV reverse transcriptase (Tiangen, Beijing, China) according to the manufacturer’s instructions. The sequences of the specific primer sets are listed in Additional file 2. The Actin (CL5382.Contig2_All) gene was used as an endogenous control, qRT-PCR was performed in 96-well plates in a Bio-Rad CFX96 real-time PCR system (Bio-Rad, CA, USA) with a SYBR Green-based PCR assay. The final volume for each reaction was 20 μL with the following components: 2 μL diluted cDNA template (1 mg/mL), 10 μL SYBR Green Mix (Bio-Rad, CA, USA), 0.4 μL forward primer (10 μM), 0.4 μL reverse primer (10 μM) and 7.2 μL ddH₂O. The reaction was conducted under the following conditions: 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 10 s and 60 °C for 30 s. The melting curve was obtained by heating the amplicon from 65 °C to 95 °C at increments of 0.5 °C per 5 s. Each qRT-PCR analysis was performed with three biological replicates. The relative quantification of gene expression was computed using the 2^-ΔΔCt method.