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Old 08-07-2017, 08:44 AM   #1
candida utilis
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Default 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?

Thanks
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Old 08-07-2017, 10:33 AM   #2
cmbetts
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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.
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Old 08-08-2017, 09:37 AM   #3
jteeee2
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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). http://arcticzymes.com/technology/kits/heat-and-run/

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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