Additional file 8.
A flow chart of DSN-mediated (duplex-specific nuclease) suppression subtractive hybridization (SSH). A small amount of RNA samples from tester (GM077) and driver (BP034) was used for template-switching cDNA synthesis and step-out PCR amplification . SP6 and T7 RNA polymerases were then employed to generate sufficient tester and driver transcripts, respectively. After a secondary reverse transcription and RNA digestion, the tester cDNAs were subjected to an excess amount of driver RNA for hybridization. Hybridization was performed by denaturation and ressociation. cDNAs in hybrids with RNA were digested by duplex-specific nuclease. The left-over single-stranded cDNAs from hybridization were only the temples for exponential PCR amplification to generate cDNA fragments for construction of a cDNA library. Tsp (template-switching primer), 3’ap (adaptor primer), PI (primer I).
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Zhang et al. BMC Plant Biology 2012 12:230 doi:10.1186/1471-2229-12-230