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Why are nuclei sorted at the end of the Frankenstein protocol?

Posted By: janette60, on Jul 12, 2019 at 1:49 PM

Is flow sorting necessary if using nuclei for 3'RNA-seq?

Thank you

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Re: Why are nuclei sorted at the end of the Frankenstein protocol?

Posted By: Luciano2018, on Sep 20, 2019 at 1:32 PM
I would definitely recommend it. The data is much cleaner. Sorting has two (main) advantages:

-allows you to see the ploidy distribution in your sample, and you may learn a priori whether your sample is diploid or polyploid or a mix of both before data analysis. You can select what population (based in ploidy) want to profile. This is great info for tumour samples.
-removes debris, doublets and floating RNAs and reduce potential RT contaminants because each nuclei comes in 1 nL (re 75-80 micron nozzle) of sheath Buffer (+some PBS/BSA/RNAse inhibitor from the sample). So you are not adding random stuff. You just need to be mindful of the numbers you sort and consider most sorters are off between 30-40% so sort more to compensate for that.