GSM2344491: Tomato_Control (PARE); Solanum lycopersicum; RNA-Seq
Total RNA from the leaf samples was extracted using the mirVana miRNA isolation kit (Ambion, Austin, TX, USA), with modifications. Briefly, 100 mg of leaf sample was ground with 800 µl of Lysis/Binding buffer. Then, 80 µl of miRNA homogenate was added and the resulting solution was mixed by vortexing.  RNA was separated by acid phenol-chloroform extraction followed by DNase RQ1 (Promega, MA) treatment. RNA integrity was examined in a 2100 Bioanalyzer (Agilent Technologies, California, USA). Total RNA obtained from 21-days post inoculation (dpi) plants were subjected for mRNA purification using GenElute mRNA Mini Prep kit according to the manufacturers instructions (Sigma-Aldrich). An RNA adapter-containing MmeI restriction endonuclease site, 5’-PARE RNA adapter, was ligated to the 5’ uncapped ends of the purified mRNA by incubating at 37ºC for 1 h in the presence of T4 RNA ligase I (New England Biolabs, Inc, MA, USA). The unligated adaptor was removed with GenElute mRNA Mini Prep kit. Obtained RNA products were subjected to cDNA synthesise using Target RT-primer followed by 7 cycles of PCR amplification using 5’-cDNA PCR primer and 3’-cDNA PCR primer. The amplicons were subjected to MmeI restriction endonuclease digestion for 1 h according to the manufacturers instructions (New England Biolabs Inc, MA, USA). The products of were separated on 5% polyacrylamide gel and then the nucleic acid band of approximately 70-bp was eluted using elution buffer (10 mM magnesium acetate tetrahydrate, 0.5 M ammonium acetate, 1 mM EDTA [pH 8.0], 0.1% [w/v] SDS). The eluted products were subjected to 15 cycles of PCR amplification using Illumina universal primer along with Indexed TruSeq 3’ index primers.