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candida utilis 08-07-2017 07:44 AM

DNase treatment for SMART Seq2
I notice that DNase I treatment is not included in the SMART Seq2 protocol. May I know how efficient Oligo-dT30VN will bind to mRNA only? Is there any possibility that Oligo-dT30VN can also amplify DNA from genomic DNA, if there is no DNase I treatment?


cmbetts 08-07-2017 09:33 AM

There's a couple reasons not to worry about it, but the primary ones are:
1) The denaturation temperature before RT isn't high enough to melt high MW gDNA
2) Even if gDNA was primed, the template switching mechanism requires the RT to hit a 5' end and neither the RT nor the PCR polymerase are anywhere processive enough to worry about capturing whole chromosomes.

jteeee2 08-08-2017 08:37 AM

If you're really interested DNase treating, we have had good success incorporating this product into the front end of several RNA Seq protocols (including SmartSeq).

We have seen that significant DNA contamination can affect the quality of the SmartSeq library, so I think it's definitely worth considering.

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