RNA-Seq of watermelon leaf: Kaimel cultivar under control replicate 1
Total RNA was extracted from watermelon leaf samples using standard methods. mRNA was purified using oligo-dT attached magnetic beads. Following purification, the mRNA was fragmented using fragmentation buffer at elevated temperature. First strand cDNA was synthesized using random hexamer primers and reverse transcriptase. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, adapters with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 300 bp in length, the library fragments were purified with AMPure XP system. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 5300 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina NovaSeq platform and paired-end reads were generated.