Procedure used, from tissue sampling to sequencing. Two genotypes (G1 and G2) were subjected to two watering regimes (IR and NI). Shoot apices from three trees per genotype and per treatment were collected in the field. Total RNA was extracted from three apices per tree. Two replicate RNA extractions were carried out for the construction of two independent replicate cDNA libraries per genotype and per treatment (resulting in 8 templates for cDNA library construction). For sequencing, each cDNA library was tagged, and two independent multiplexes were created by pooling one sample for each genotype and treatment combination. Multiplex #1 was sequenced with two 454-Roche FLX Titanium half-runs, resulting in eight sequencing sets, whereas multiplex #2 was sequenced with one half-run and resulted in four sequencing sets.
Villar et al. BMC Genomics 2011 12:538 doi:10.1186/1471-2164-12-538