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Can anyone offer any insight why in SMART-seq2 the RT and TSO primers are designed in a way that cDNA is amplified by semi-suppressive PCR (suggesting that without it, there would be a lot of primer-dimer products amplified) whereas in SMART-seq3/FLASH-seq, the RT/TSO primers carry different anchor sequences for cDNA amplification which does not rely anymore on semi-suppressive nature. So is the primer-dimer formation suppressed by either different RT enzyme usage or reaction buffer conditions in SS3/FLASH-seq or somehow these new RT/TSO primers do not form primer-dimers as efficiently as those in SMART-seq2? Is there then any RT/TSO design principle that one should follow to avoid excess formation of primer-dimers during template-switching RT? Thanks a lot!
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