Hello. Has anyone tried Illumina's DSN normalization protocol. We're planning to have a go using a couple of FFPE samples. Looking through the methods it seems that tight temperature control is pretty important, but I would appreciate any input from people who know any of the little tricks that never make it into the official methods.
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Use flip cap strip tubes this decreases the time getting them open and thus reducing the chance you lose your Cot curve.
Keep everything close and use two pipettors at a time (one in each hand), one to add the buffer or enzyme and the other to mix. This will reduce the time you have the lid off. You really want to move fast but do not want to introduce bubbles.
I think the protocol says to mix with a pipettor set at 40 ul but I do 35 ul since the 40 always introduces bubbles for me and if you remove the tube from the thermal cycler its game over. -
Thanks a lot, that's exactly the sort of thing I was after. It's too expensive an experiment to be messing around for ages trying to get a pilot study working due to silly beginners' mistakes.Comment
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Hi,
all the posts here on DSN are rather old and likewise publication using the method are few and date a couple of years back.
Thus, I was wondering if people are still using DSN normalization for RNAseq libraries or is it just not worth the trouble?
Specifically I am interested in DSN treatment coupled with poly A selection in human samples in order to be better able to identify lowly expressed transcripts.
Any suggestions or comments appreciated,
thanks,
blancoComment
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Hello,
Previously DSN normalization has been expensive as the critical component (DSN) is costly. I currently developed a duplex-specific nuclease for our company as an alternative to the current DSN on the market (made in the USA), PureRec Duplex-Specific Nuclease that is more specific for digesting dsDNA and DNA in DNA-RNA hybrids. Please check out Zymo Research (E1020S, E1020-200, and E1020-1000). It is a fraction of the cost and recombinant so you can perform multiple experiments for RNA seq libraries. It is available for purchase and also we have free samples available. Hope this helps lower the cost for you to do your work!Comment
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