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Re: single nuclei RNAseq from frozen tissues

Posted By: SaraFH88, on Jan 24, 2019 at 2:40 AM

Hi Luciano

 

Thank you so much for sharing your protocol and helping out the single cell/nuclei community!

 

I have a quick question about the cell strainer: the 10X nuclei protocol suggests a 40μm whereas your protocol use a 35μm. Did a smaller size mesh improved purity without compromising the amount of isolated single nuclei? Would a 40μm also work?

 

Thank you very much for all your help.

Cheers,

Sara

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Re: single nuclei RNAseq from frozen tissues

Posted By: Luciano2018, on Jan 25, 2019 at 10:09 AM
Hi Sara,

35 or 40 um work similarly, no difference I can notice. I do do a pre filter with 70 um strainer before first centrifugation to leave all the undigested tissue followed for the 35-40 um strainer before sorting.

I hope this helps!

Re: single nuclei RNAseq from frozen tissues

Posted By: SaraFH88, on Feb 15, 2019 at 2:45 AM

Hi Luciano

 

Your protocol works wonderfully and it is a great addition to the 10X protocol profile.

I have just one last question regarding the FACS sorting of nuclei. Is there a specific method you used to separate the nuclei signal from the FACS background noise? Or used a special flow cytometer device?

I appreciate all the help you have already given me.

Thank you.

Cheers

Sara

Re: single nuclei RNAseq from frozen tissues

Posted By: Luciano2018, on Feb 15, 2019 at 7:16 AM
Hello Sara,
I’m glad it also works for you! Yes, I use DAPI to stain the nuclei and there is very little if any background at all. We have used several different sitters, two that work nicely are the Facsaria 2 and the Fusion.

What type of background are you observing?