Tianscript 2 rt kit

Total RNAs were extracted by Trizol (Invitrogen, USA). TIANScript II RT Kit (Tiangen, Beijing) was used to reverse transcribe RNAs into cDNAs. RT-qPCR reactions were prepared using the RealMastcrMix (SYBR Green, Tianjian Biotechnology Co., Ltd., Beijing, China). The relative expression levels of LINC00261 and TNRC6A were normalized to the internal reference GAPDH using the 2^-ΔΔCt method, while the expression levels of miR-522-3p were normalized to U6-snRNA. The primer sequences used in this study are listed in Table 1.

Total RNA was extracted by using RNAiso Plus (9109, Takara, Kusatsu, Japan) and a Direct-zol RNA MicroPrep kit (R2060, Zymo Research, California, USA), and the concentration was measured with a Nano-300 ultramicro spectrophotometer (Allsheng, Hangzhou, China). cDNA was obtained according to the instructions of the TIANScript II RT kit (KR107, TIANGEN, Beijing, China). The expression of transcripts of the target gene was measured by using a Light Cycle® 96 instrument (Roche) with Hieff UNICON SYBR green master mix (11198ES08, Yeasen, Shanghai, China). AS analyses were performed on a RePure-A PCR instrument (BIO-GENER, Hangzhou,China). Primers were synthesized by Sangon Biotech (Additional file 6: Table 3). The expression level of Gapdh or Vasa was used as the control, and this value was set as 1. The relative transcript expression levels of other samples were obtained by comparing them with the control results.

Approximately 1 µg of high quality RNA per sample was extracted using plant RNA extraction kits (TIANGEN, China). The first strand of cDNA synthesis was performed using the TIANScript II RT kit (TIANGEN, China). We diluted 20 µL of cDNA from each sample to a total volume of 200 µL using DEPC water. These were used as qPCR templates. The reaction mixture contained1 µL cDNA, 0.5 µL each of the forward- and reverse-specific primer, 10 µL chamQ SYBR qPCR Master Mix (Vazyme, China), and 8 µL DEPC water, for a total volume 20 µL. The qPCR reaction was conducted in an ABI 7300 Real-Time PCR system with the following amplification program: 95 °C for 5 min, 40 cycles of 95 °C for 5 s, and 60 °C for 35 s. The pomegranate PgActin gene served as the reference gene, and the relative expressions levels of the genes were calculated according to Livak & Schmitten (2001) (link). Each sample was quantified in triplicate. Data were analyzed using SPSS software (22.0, USA) and Excel. All primers used for qPCR assay are shown in Data S2.

For each RNA sample, 4 μg of total RNA was reverse-transcribed using the TIANScript II RT Kit (Tiangen, Beijing, China). The cDNA was diluted 1:10, and 2 μl of the cDNA was used as template in 20 μl of amplification reaction, using SYBR ^® Premix Ex Taq^™ II according to the manufacturer’s protocols (Takara, Dalian, China) on a Mastercycler ep realplex (Eppendorf, Hamburg, Germany). The specific primers for each gene were designed using Beacon designer 7.5 software (Palo Alto, CA, USA), and are listed in Table 4.

Approximately 5.0 g or 300 μL of each sample was homogenized separately in phosphate-buffered saline (PBS). According to the manufacturer’s instructions, viral RNA was extracted using the Viral RNA Extraction Kit (Takara, Dalian, China) and then the TIANScriptII RT Kit (TIANGEN BIOTECH (BEIJING) CO., LTD., Beijing, China) was used to acquire cDNA through reverse transcription. The primers of Nsp2 and ORF5 were designed based on the alignment of published PRRSV genome sequences obtained from the NCBI GenBank database (Supplementary Table S2). The cDNA was used for the PCR amplification of Nsp2 and ORF5. Then, the amplicons for each virus were submitted to Sangon Biotech Shanghai Co., Ltd., Shanghai, China for sequencing. The nucleotide sequences were determined for at least three independent cDNA clones.

Total RNA was extracted using a Quick RNA Isolation Kit (Waryoung, Beijing, China) and cDNAs were synthesized using TianScript II RT Kit (Tiangen Biotech, China). The qRT-PCR analysis was performed using ABI PRISM 7500 Real-Time PCR System (Applied Biosystems). The cucumber UBIQUITIN (Csa000874) and Arabidopsis ACTIN2 (AT3G18780) genes were used as internal controls. Three biological replicates were performed. Sequences of all the primers used in this study were shown in Table S2.
For gene expression pattern analysis, the cucumber root, stem, leaf, tip and tendril of 16-day-old seedlings, axillary bud from the top of the lateral branch, male and female buds at the 10^th stage of flower development, male and female flowers at anthesis, 1.5 cm fruit, 2 cm fruit and fruit on the day of blooming were harvested for RNA isolation.
For analysis of ABA and auxin-induced gene expression, the two-week-old cucumber seedlings were treated with 100 μM abscisic acid (ABA) or 100 μM indole-3-acetic acid (IAA). The seedling tips were collected at 0, 0.5, 1, 3, 6, 9, 12 and 24 h after treatment.

Ten randomly selected genes were performed qRT-PCR. One microgram of total RNA was transcribed into cDNA using a TIANScript II RT Kit (TIANGEN, Beijing, China) and qRT-PCR was performed using the OneStep Real-Time System (Applied Biosystem, Carisbad, CA, USA), according to the manufacturer’s instructions. The reference gene actin was used for normalization, and three independent biological replicates were used for each sample. The comparative CT method (2^−ΔΔCT method) [28 (link)] was used to analyze the genes expression levels. The ten randomly selected gene IDs were G26028, G48802, G24647, G19689, G22719, G24812, G16071, G44971, G3829, and MSTRG.52707. The specific primers used are listed in Table S18.