title: Noccaea caerulescens transcriptome sequencing
design: Poly(A)+ RNA isolated from roots, shoots flowers were used for cDNA synthesis. First strand cDNA synthesis was primed using random hexamer primers. After ligation of Roche/454 adapters A and B to the 5'- and 3'-ends, the cDNA was amplified using 12 PCR cycles and proofreading DNA polymerase.   Normalization was performed by denaturation and re-association of cDNA. A hydroxylapatite column was used for separating Re-associated double strand - from single strand cDNA. Single strand DNA was amplified using six PCR cycles. After size selection on agarose gel, a final normalized cDNA library was obtained consisting of double stranded fragments between 500 – 850 bp with the following structure: 5’-454-Adapter A (CCA-TCT-CAT-CCC-TGC-GTG-TCT-CCG-ACT-CAG),  5’- barcode (CACACG),  5’ adapter (GAC-CTT-GGC-TGT-CAC-TCA-GTT), cDNA insert (400-750bp), 3’ adapter (TCG-CAG-TGA-GTG-ACA-GGC-CA), 3’-454-Adapter B (CTG-AGA-CTG-CCA-AGG-CAC-ACA-GGG-GAT-AGG). Prior to Roche/454 sequencing, cDNA library molecules were clonally amplified using a two copies per bead ratio for one Large Volume Emulsion PCR, following the manufacturer’s protocol (Roche, Genome Sequencer FLX Titanium Series).