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‘Frankenstein’ protocol for nuclei isolation from fresh and frozen tissue-version 3 kit

Posted By: hmm10, on May 7, 2019 at 7:43 AM

Has anyone used this protocol with version 3? If so, did you make any changes? I'm interested in the sort into RT buffer because the volume has changed between Version 2 and 3.

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Re: ‘Frankenstein’ protocol for nuclei isolation from fresh and frozen tissue-version 3 kit

Posted By: Luciano2018, on Sep 20, 2019 at 1:41 PM
Yes, for v3 use as follows:

RT Buffer 20 uL
Reducing Agent B 2 uL
TSO primer 3.1 uL
(Don’t add the RT enzyme C yet)
Then you have 46.6 uL. If using a 75-80 micron nozzle each nuclei will come in approximately 1 uL (you may need to determine this for your sorter of choice as this varies). If you plan to sort 20 K nuclei then they will come in ~20 uL, therefore to the RT mix add 20.6 uL of RNAse free water or 1x PBS (no Ca/Mg2+). You can add less if unsure to leave more dead volume.

After sorting, measure the volume in the tube, add water or 1x PBS to a final volume of ~71.7 uL (small difference in volume make no changes whatsoever, so don’t waste too much time or stress out getting this right but mostly getting this done fast) and add 8.3 uL of RT Enzyme C, mix gently 5-6 times and load.

Good luck!

Re: ‘Frankenstein’ protocol for nuclei isolation from fresh and frozen tissue-version 3 kit

Posted By: eseifie, on Oct 15, 2019 at 9:46 AM

Hi, 

 

I am new to 10X please how can I get access to the Frankenstein protocol.

My email is eseifie@yahoo.com

 

Many Thanks,

Ese

Re: ‘Frankenstein’ protocol for nuclei isolation from fresh and frozen tissue-version 3 kit

Posted By: shauna-10x, on Oct 15, 2019 at 11:03 AM

Hi Ese,

 

You can download the protocol here: https://community.10xgenomics.com/t5/Customer-Developed-Protocols/ct-p/customer-protocols

 

Scroll down to the bottom of the page to find the Frankenstein protocol. 

 

Best,

Shauna

Re: ‘Frankenstein’ protocol for nuclei isolation from fresh and frozen tissue-version 3 kit

Posted By: Luciano2018, on Oct 15, 2019 at 1:50 PM