See Additional file 11: Tobacco EST generation.doc for description of the construction and processing of the cDNA libraries.
It might be not sampled due to technical issues related to the processing of RNA and cDNA before sequencing.
Processing of mRNA to cDNA, labelling, hybridization to A. niger Affymetrix GeneChips (Affymetrix, Inc., Santa Clara, CA) and fluorescence scanning were performed at the Nottingham Arabidopsis Stock Center NASCC, Loughborough, UK) Affymetrix facility.
The direct processing of the first strand cDNA enhances the cloning of "difficult" target sequences.
Processing of total RNA included synthesis of cDNA (One-Cycle cDNA synthesis kit; Affymetrix, Santa Clara, CA, USA), biotin labeling (3′IVT Labeling Kit Affymetrixx), hybridization, and scanning according to standard Affymetrix protocols.
All steps of microarray processing (cDNA production, cDNA labeling, microarray hybridization, data quantification, data normalization using LOWESS) were carried out by the TIGR Expression Profiling Service [ 15].
Processing of the data from 108 spotted cDNA microarrays included background subtraction and log2-transformation of fluorescence intensities, and removal of flagged spots or spots that did not surpass a minimum intensity threshold of 200.
Our work highlights the critical role that the method of cDNA priming can play in determining what transcripts and processing activities can be measured.
Finally, signal processing was performed using vector oligonucleotide data to correct the relative amount of cDNA present in each spot.
We focus on data processing effects that need to be considered not only in the case of cDNA arrays but also for other types of arrays (here only shown exemplarily for Affymetrix GeneChips).
