Omni plant rna kit dnase 1

Total RNA samples were extracted using the omni-plant RNA kit (DNase I) (CoWin Biosciences, Beijing) following the provided protocol. RNA quality and quantity were assessed using a NanoDrop 2000, an Agilent2100 nano, and were checked by RNase-free agarose gel electrophoresis.
The libraries were prepared by Illumina TruSeq™ RNA sample prep kit technology. The complete mRNA was obtained by the enrichment of magnetic beads with oligo (dT), and then the mRNA was randomly broken by adding fragmentation buffer. The small fragments of 300 bp were screened out by the magnetic beads. First-strand cDNA was synthesized using random hexamer primer and reverse transcriptase, and then the second strand was synthesized. End repair mix was added to make the sticky end of the cDNA into a flat end, and then a “A” base was added to the 3′ end.
The libraries were sequenced on an Illumina NovaSeq 6000 platform according to manufactures’ instructions. After sequencing, software seqprep and sickle were used to remove the linker sequence, low quality reads, any anonymous nucleotides greater than 10%, and adapter to obtain clean data. The clean data was assembly using trinity software (Grabherr et al., 2011 (link)). The software transrate and busco were used to evaluate and optimize the assembly results. Finally, we obtained the unigenes database by aggregating the assembled sequences.

Total RNA was extracted using the OmniPlant RNA Kit (DNase I) (CoWin Biosciences, Taizhou, China). The first strand of cDNA was synthesized from 15 μL of total RNA by using MonScript™ RTIII All-in-One Mix with dsDNase (Monad Biotechnology Co., Ltd., Wuhan, China). RhUBI2 (JK618216) and Actin were used as internal controls [40 (link)]. Primers were designed using Primer Premier 5.0 software (Premier Biosoft International, Palo Alto, CA, USA) and synthesized by Sangon Biotech (Shanghai, China) (Table S2). qRT-PCR was performed on a ABI 7500 FAST DX Real-Time PCR instrument (Thermo Fisher Scientific, Inc., Waltham, USA). Each reaction was conducted in a 20 μL mixture containing 10 μL of 2× Universal Blue SYBR Green qPCR Master Mix (Wuhan Servicebio Biotechnology Co., Ltd., Wuhan, China), 7.4 μL of RNase-free H₂O, 1 μL of cDNA, 0.8 μL of forward primer, and 0.8 μL of reverse primer. The PCR machine was programmed as follows: PCR initial activation step for 2 min at 95 °C, followed by 40 cycles at 95 °C for 5 s and 60 °C for 30 s. The relative gene expression was calculated using the 2^−ΔΔCT method [41 (link)].

The total RNA was isolated from 100 mg of cassava leaves by using the OmniPlant RNA Kit (DNase I) (ComWin Biotech Co., Ltd, China). For isolated RNA, the quality was controlled through agarose gel electrophoresis and by using the NanoDrop 2000 (Thermo, Waltham, MA, USA), and the concentration was determined by using the NanoDrop 2000. The first-strand cDNA synthesis was conducted with 1 μg of quality-controlled RNA by using the PrimeScript^™ RT reagent Kit with gDNA Eraser [TaKaRa Biomedical Technology (Beijing), China]. Then, the synthesized cDNA product was diluted 10 times with RNA-free water for further use.