title: Reads from leaves of plants subjected to salt-stress (Source 4)
design: cDNA synthesis was performed with 3.5 µg of total RNA using Message Amp-II kit (Ambion) following the experimental protocol, as instructed. Briefly, first strand cDNA synthesis was primed with T7 Oligo(dT) primers. After a second strand cDNA synthesis reaction, 10–12 ng of synthesized double-stranded cDNA were amplified by in vitro transcription and the resulting 70–90 µg of antisense RNA (aRNA) was purified using Qiagen RNAeasy columns (Qiagen). A second round of cDNA synthesis was performed using the aRNA as template. First and second strand cDNA synthesis were as described above except that random primers (mostly hexamers) were used at the first strand synthesis. This procedure yielded about 10 µg of cDNA that were purified using the DNA Clear Kit for cDNA purification (Ambion). cDNA was then treated with the Ampure magnetic particles (Agencourt, Beckman Coulter) to obtain fragments of 200 - 700 pb before sequencing.