Accurt genomic dna removal

The total RNA was extracted from the leaves of chickpea cultivar Dwelley using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) from 100 mg samples in accordance with the manufacturer’s protocol. First strand cDNA synthesis and genomic DNA elimination were performed simultaneously using 5X All-In-One RT MasterMix, containing AccurT Genomic DNA Removal (Applied Biological Materials Inc., Richmond, Canada). cDNA samples were stored at−80 °C until use. Full-length ORFs were amplified with Phusion^® High-Fidelity DNA Polymerase (NEB, Ipswich, MA, United States) using gene-specific primer pairs (Supplementary Table S1) using the following protocol: initial denaturation at 98°C for 30 s and 35 cycles of 98°C for 10 s, 60°C and 72°C for 30 s each and followed by a final elongation at 72°C for 8 min. Amplified PCR products with appropriate expected sizes were purified with the Monarch^® DNA Gel Extraction Kit (NEB). Purified PCR products were cloned into the pMiniT 2.0 vector (NEB) and transformed into DH10B high-efficiency E. coli competent cells (NEB). The plasmids were recovered from E. coli using PureYield™ Plasmid Miniprep (Promega, Madison, WI, United States), verified using Sanger sequencing (Laboratory of Biotechnology & Bioanalysis, Pullman, WA, United States), and compared to the GenBank sequences of CaPGIP1 (XM_004504675), CaPGIP3 (XM_004493500), and CaPGIP4 (XM_012713804).

Total RNA was extracted using the Quick RNA plant isolation kit (Beijing Yueyang Biotechnology Ltd., Beijing, China), and cDNA was synthesized using the OneScript^® cDNA Synthesis kit with AccuRT Genomic DNA Removal (Applied Biological Materials Inc, Vancouver, Canada). qRT-PCR was performed in 96-well plates with an Applied Biosystems 7500 real-time PCR system (Applied Biosystems, Foster city, CA, USA) using the One-Step BrightGreen qRT-PCR-Low ROX kit (Applied Biological Materials Inc, Vancouver, Canada). Primers were designed using Primer3Plus, and primer specificity was evaluated with NCBI primer-BLAST against the cucumber genome. The specificity of the amplified product was confirmed by a melting curve. Three biological replicates and 3 technical replicates were performed for each combination of cDNA samples and primer pairs. The cucumber β-actin served as the internal control gene [101 (link)]. The relative expression of target genes was analyzed by comparative CT method [102 (link)]. The cucumber chitinase qRT-PCR primers are listed in Table S8.