RNAseq of single nuclei

Status: Delivered
Posted By: carlo, on Nov 16, 2016 at 1:42 AM

I would like to be able to do RNAseq from single nuclei. As I am working with frozen human brain samples, getting single cells is not feasible, while single nuclei can be recovered efficiently. We can do single nucleus RNAseq by FACS sorting single nuclei in 96 well plates and then doing SmartSeq2, but using the Chromium for single nuclei work will be a better option.

 

Comments
Posted By: achamess, on Dec 1, 2016 at 6:35 AM

Hi. I want to do the same. I've done bulk neuronal nuclei and singles using Smart-Seq2. I spoke to a rep from 10x about a month ago and he said that the issue with nuclei (in their hands) was that lysis was ineffective. It seems that nuclear membranes are more impervious to lysis than cell membranes. This doesn't sound insurmountable, but it's a problem. Also, the amount of poly-A mRNA in a nucleus is less than a cell, and so capture on the GEMs may be less effective. It's worth a try, but will cost for R&D to get it right. I hope 10x supports this application eventually, since many people are interested in nuclei, especially in neurobiology.  

Posted By: XianA, on Jan 12, 2017 at 12:12 PM

Yes, add me to this as well. It's known that studying nuclei is a good alternative to single cell on difficult tissues or cell types. We're even willing to do some R&D work as the consumer. However we're limitied by the high cost of per channel reagents.

Posted By: carlo, on Mar 17, 2017 at 3:28 AM

Following up on this idea: there is a preprint demonstrating a modification of the Drop-seq protocol for single nuclei RNAseq

http://biorxiv.org/content/early/2017/03/09/115196

 

So, it should be possible to develop dedicated single cell chips for nuclei work specifically, with appropriate microfluidic design. The correct pressure for the new chip would be encoded in a dedicated Chromium script. Of course, appropriate changes to chemistry for lysis/bead swelling for nuclei. Chemicals and chips could be packaged as a specific Chromium Single Nuclei 3' kit.

 

Will be happy to be alpha/beta tester! Smiley Wink

Posted By: shauna-10x, on Sep 20, 2017 at 8:19 AM
Status: Delivered

Hi All,

 

Thanks for your feedback and suggestions. If you haven't seen it already we posted a new demonstrated protocol for the isolation of nuclei for single cell RNA-seq. You can download it here: https://support.10xgenomics.com/single-cell-gene-expression/sample-prep/doc/demonstrated-protocol-is...

 

We also recently had an online 10x-pert Workshop for single cell sample preparation techniques and highlighted the nuclei isolation protocol.  A recording of the session can be viewed here: https://community.10xgenomics.com/t5/10x-Blog/Single-Cell-RNA-seq-Sample-Preparation-Techniques-and-...

 

Thanks,

Shauna

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