RNA-Seq of total RNA from virus infected passion fruit: leaf sample
Poly A containing mRNA molecules from diluted total RNA (1g) were purified using poly-T oligo attached magnetic beads. The purified poly-A RNA was then fragmented and primed for 1st and 2nd strand cDNA synthesis. After cDNA sythensis, the 3' ends were adenylated by addition of a single 'A' nucleotide to the 3' ends. Indexing adapters were then ligated to the ends of the ds cDNA followed by enrichment of DNA fragments by PCR with adapter molecules on both ends. This was followed by checking the quality (validation) of enriched DNA templates by loading 1l of re-suspended construct on a 2100 Bioanalyzer using an Agilent DNA 1000 chip. A final product with a band of 260bp indicated the right size and purity of the sample. After library validation, the indexed DNA library was then normalized to 10nM using Tris-HCl 10 mM, pH8.5 with 0.1% Tween 20 and sequenced.