Smartseq Exonuclease

[wyue]

Hi all,

We have a few questions about using exonuclease before the preamp step of the Smartseq protocol.

1. Is this step intended for removing primer dimers, not excess single-stranded primers? Is that why lambda exonuclease (which works on dsDNA) is chosen instead of ExoI?

2. Wouldn't lambda exonuclease digest away the double-stranded cDNA template? If the ends got digested, the priming sites may be lost.

3. We have tried replacing lambda exonuclease with ExoI for a 30 min incubation, then an 85C, 15 min inactivation before adding primers for preamp step. However, yield is very low. Can somebody explain why?

4. Does anyone have the reference for the first suggestion of using lambda exonuclease?

Many thanks!

[lovescience]

Hi, have you got answers? I am also curious about this.

[wyue]

I am afraid we don't have answers to those questions. We tried including lambda exonuclease or ExoI and the yield was low in both cases. We ended up skipping exonuclease digestion altogether and the yield was good. If you do need to include an exonuclease step, I would suggest reaching out to the authors of: https://www.nature.com/articles/s41467-018-05347-6. They mentioned the use of ExoI.