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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

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?

Re: single nuclei RNAseq from frozen tissues

Posted By: nikatag, on Aug 22, 2019 at 5:38 PM

Hi Luciano, 

We follow this protocol step by step forOCT frozen heart tissue to isolate nuclei.

And We just got back some data from the nuclei test run and would love to get your opinion on the results if possible. We are still seeing low fraction reads in cells (68%) and are wondering if that's normal for nuclei? Have you ever encountered with this type of issue? 

Also we only could isolate 2,000 nuclei out of targeted 10k. Could it possibly mean we overlysed the nuclei? or should we force cellranger mapping to call the nuclei with given lower UMI.

[ How many UMI count you set up to call it nuclei? (Usually for cells are 500 counts) ]

for ease of communication, can we set up a skype to discuss furthure? 

My email is ntaghdir@eng.ucsd.edu 

 

I really appriciate your help