Veriti 96 well thermal cycler

For the isolation of total RNA, 50–100 mg of freezing sample was ground with liquid nitrogen in a pre-treated RNase free mortar. Total RNA was extracted from the ground sample with the classical Trizol method as we described in a previous article (Yang et al., 2019 (link)). The obtained total RNA was dissolved in 30–50 μl of RNase free water. The concentration and purity of the RNA were determined using a nanodrop UV spectrophotometer. In general, RNA was quantified for the following cDNA synthesis when the value of A260/A280 was above 1.90. cDNA was produced in an ABI Veriti 96 Well Thermal Cycler (Waltham, MA, United States) using FastQuant RT Kit from Tiangen Biotech Co., LTD. (Beijing, China). Real-time PCR was performed in an ABI QuantStudio3 PCR System (Waltham, MA, United States) using SYBR Green qPCR Master Mix and gene specific primers synthesized by Sangon Biotech (Shanghai, China). The program was set as following: initial denaturation at 95°C for 10 min followed by 40 cycles of 95°C for 15 s, 58°C for 30 s and 68°C for 60 s. The primers used in this study were listed in Table 1. The relative expression of target mRNA was calculated by normalizing target mRNA C_ts to the housekeeping gene GAPDH (method of 2^-DDCt) (Yang et al., 2019 (link); Yin et al., 2019 (link)). Undetectable of the target mRNA was defined as the C_t value greater than 30 cycles.

To verify the novel transcribed regions, we randomly selected 18 such regions to perform RT-PCR. ACT2 gene (AT3G18780) was used as a positive control, for which primers were designed on two neighboring exons. Total RNAs of Col-0 inflorescences were obtained by ZYMO RESEARCH ZR Plant RNA Miniprep™, and reverse transcription was carried out by TAKARA PrimeScript™ RT Master Mix. Forty cycles were run on ABI Veriti® 96-well Thermal Cycler with Tiangen Taq DNA Polymerase (ET101) and proper primers. The same cDNA library was also used for real-time PCR validation of genes with different expression values. The real-time PCR was performed with TAKARA SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) on ABI StepOnePlus™ Real Time system. Primers for these experiments are listed in Supplementary Table 7.

Total RNAs were isolated from cells using TRIzol reagent (SparkJade, Qingdao, China) according to the manufacturer's instructions. The concentration and purity of total RNAs were determined spectrophotometrically by measuring the absorbance at 260 nm and 280 nm using a UV spectrophotometer, and cDNA was produced using an ABI Veriti 96‐Well Thermal Cycler (Waltham, MA, USA) and FastQuant RT Kit with gDNase (Tiangen). Real‐time PCR was performed in an ABI QuantStudio3 PCR System (Waltham, MA, USA) using SYBR Green qPCR Master Mix and gene‐specific primers with an initial denaturation step at 95°C for 10 minutes followed by 40 cycles of 95°C for 15 seconds, 58°C for 30 seconds and 68°C for 60 seconds. The primers for qRT‐PCR are listed in Table 1. The fold change in the expression of targets relative to the housekeeping gene GAPDH was calculated based on the 2^‐ΔΔCt relative expression formula.