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26 Replies

Re: single nuclei RNAseq from frozen tissues

Posted By: jens-10x, on Mar 8, 2018 at 8:45 PM

Hi Luciano,

 

Up to 3 mM is safe to use. We start seeing some impact on the library quality (particularly the % of mapped reads to the reference) if magnesium concentrations are significantly higher.

 

Keep us posted and feel free to contact our Support group (support@10xgenomics.com) directly if you have any more questions.

 

Jens

 

Re: single nuclei RNAseq from frozen tissues

Posted By: rmtsoa, on Mar 19, 2018 at 11:50 AM

Hi Jens:

 

I just had a conversation with my friend about the snRNAseq. From what he told me, there might be some issue of inefficient nuclei lysing after nuclei capturing for RT reaction. Do you have any internal analysis to show the lysing efficiency in the droplet? 

 

Thanks,

Rosemarie 

Re: single nuclei RNAseq from frozen tissues

Posted By: almisharin, on May 25, 2018 at 3:38 PM

Would love to hear updates from Luciano2018 and response to rmtsoa. 

Luciano, hope you solved clumping problem. Were you able to get meaningful libraries from the single nuclei suspension?

Rosemary: did you get any response from 10x?

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Re: single nuclei RNAseq from frozen tissues

Posted By: Luciano2018, on May 25, 2018 at 4:13 PM
Hi guys,

Yes! I tried a few things and made a “Frankenstein” protocols (bits a pieces from different protocols out there) and now nuclei prep works very well. Libraries look quite nice, and sequening data too. Still have a bit of background RNA I’m guessing, probably normal but since the UMI counts in nuclei is lower than in cells the background rna is more significant. Still, I get good cut off for GEMs with nuclei versus empty GEMs. SoupX will make this background go away.
Thanks for reaching out!

Re: single nuclei RNAseq from frozen tissues

Posted By: achamess, on May 30, 2018 at 4:42 PM

Luciano2018 - I'm glad you got nuclei working. Can you say more about the buffers you used, how you homogenized (dounce?) and whether you needed to perform a density gradient (Optiprep)?