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Old 03-14-2012, 12:18 AM   #1
Simone78
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Default why rG and not dG in the template switch oligos (TSO) in single cell library preps?

Hi all,
I am preparing single cell libraries for RNAseq using the SMARTer II kit (Clontech) and I was wondering whether there is a reason why they use rG in the template switch oligo (TSO).
They start from total RNA and do a regular RT using oligo-dT primers to make the first strand of cDNA. When the reverse transcriptase reaches the 5′ end of the mRNA template, it preferentially adds 2-5 cytosines. The template-switching oligo, which has 2-5 guanosines, anneals transiently, and reverse transcriptase then switches template and synthesizes the complementary strand. But why do they need 3 rG at the 3-end of this oligo (which is otherwise a regular oligo with deoxynucleotide bases)?
It would be much cheaper and easier to use 3 dGs, but I guess the company already tried (and discarded) this option.
Thanks a lot!
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Old 03-14-2012, 09:40 AM   #2
zhengz
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RNA-DNA binging is stronger than DNA-DNA. The rG TSO would anneal better than a dG one to the short stretch of 2-5 cytosines.
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Old 03-14-2012, 10:56 AM   #3
pmiguel
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That is just what the reverse transcriptase in question prefers, right? I don't think how well the oligo binds comes into it.

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