The Rsa I-digested tester cDNA was ligated with adapters and hybridized with an excess amount of Rsa I-digested driver cDNA to subtract the cDNA population present in both tester and driver.
It thus appears that very abundant tester-specific transcripts escape normalisation, whereas very abundant transcripts expressed in both tester and driver escape both subtraction and normalisation.
Both, tester and driver, cDNA populations were processed following manufacturer instructions, with some modifications.
Both tester and driver representations were digested with Sau3AI to remove the R-Bgl adaptors.
Arabidopsis Thaliana RNAs (Stratagene, Spike RNA 201, 204, 205, 208, and 209) were added as reporter exogenous RNAs at different concentrations, either specifically in tester material or in both tester and driver materials.
Two of them (3P11 and 3I20) were tester-specific, but the others (3L22 and 3C24) were expressed in both tester and driver, with double their relative abundance in tester than in driver (3L22 and 3C24 representing respectively about 1.2 and 1.5% of morula driver cDNAs).
Very abundant transcripts common to both tester and driver may escape both normalisation and subtraction, giving rise to abundant background clones in these libraries.
Alignments were compared to determine which reads aligned better to the Tester or Line references and which reads aligned equally well to both the Tester and Line references.
This suggests that the reasons for testing and the reasons for not testing may have played a role in both testers' and non-testers' decision-making.
The cDNA under investigation was designated tester, and the reference cDNA was designated driver.
