Roots that were collected from Mirabilis himalaica at harvest period (100 days after planting)
Total RNA sampled from roots was isolated using an EASYspin Plus Plant RNA isolation kit. A 2100 Bioanalyzer was used to perform the total RNA quality control. Qualified RNA samples were digested by DNaseI for 30 mins at 37°C and purified using Dynabeads® Oligo (dT) 25. Then, 100 ng of purified mRNA was used to establish a library using a NEBNext® Ultra™ RNA Library Prep Kit for Illumina . First, random primers and First Strand Synthesis Reaction Buffer (5×) was added to disrupt the mRNA, followed by incubation at 94°C for 15 mins. The sample was set on ice immediately afterwards. The forward strand of the cDNA was synthesized by adding Murine RNase Inhibitor and ProtoScript II Reverse Transcriptase to the sample, while the reverse strand of the cDNA was synthesized by adding Second Strand Synthesis Reaction Buffer (10×) and Second Strand Synthesis Enzyme Mix to the sample. The samples were then purified by adding 1.8 times volumes of AMPure XP Beads, followed by base repairing and removal of uracil. Then, 0.6 times the volume of AMPure XP Beads was then added to the sample and mixed well and the mixture was left to stand for 5 mins, after which it was placed on a magnetic rack for 5 mins. The supernatant was then removed and 0.25 times AMPure XP Beads were added for purification. Ligated cDNA 300–500 bp in size was received. Then, the samples were amplified by 15 rounds of PCR and purified using 1-times the volume of AMPure XP Beads. The library was then received and quantified using Qubit, 2% agarose electrophoresis, and a High Sensitivity DNA chip to ensure the quality of the library. Finally, 10 ng of the library was taken, and a cluster generation was performed in cBot using TruSeq PE Cluster Kit followed by two-way sequencing on Illumina Hiseq™2500/MiSeq™.