title: Vanilla planifolia transcriptome
design: Poly A-RNA, isolated from 6-month-old vanilla pod tissue was fragmented with ultrasound. RNA first-strand cDNA synthesis was carried out using a N6 randomized primer and M-MLV RNaseH-reverse transcriptase. 454 adapters A (CCATCTCATCCCTGCGTGTCTCCGACTCAG) and B (CTGAGACTGCCAAGGCACACAGGGGATAGG) were ligated to the 5'and 3' ends of the cDNA. The cDNA was finally amplified with PCR (14 cycles) using a proof reading enzyme. The cDNA library was normalized by one cycle of denaturation and reassociation of the cDNA. Reassociated ds-cDNA was separated from the remaining ss-cDNA (normalized cDNA) by passing the mixture over a hydroxylapatite column. After hydroxylapatite chromatography, the ss-cDNA was amplified with 11 PCR cycles. The cDNA in the size range of 500 – 700 bp was eluted from a preparative agarose gel and subjected to Titanium sequencing.