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Fixed tissue dissociation and sequencing

Posted By: abaffyp, on Feb 8, 2018 at 12:13 AM

Hello,

 

I'm trying to find, if somebody try to works with fixed tissue for signle-cell preparation. We're study genes which are differentially expressed after stress like a injury. So, in my idea, first I want to have fixed tissue (i.e. in methanol), dissociate it and after that, start with single-cell preparation.

 

Do you have same experience with work of fixed tissue?

 

Thanks

 

Pavel

3 Replies

Re: Fixed tissue dissociation and sequencing

Posted By: carl, on Feb 8, 2018 at 1:31 AM

Hi Pavel,

 

We haven't done any single cell work yet but members of my lab have had some success with dissociating samples stored in RNAlater. This works best for our tissue if you use enzymatic dissociation after slicing up the tissue into ~50uM pieces using a microtome. One bottleneck we are dealing with at the moment is preserving RNA quality during the dissociation process - it is in perfect condition if you directly lyse the sample but suffers from the presence of the microtome mountant in the dissociation buffer and is only partially rescued by presence of RNase inhibitors.

 

Other similar preservatives are available and we have heard good things about the suitability for single cell of a new product called RNAassist but it is not available on the market yet and they don't respond to contact requests through their website...

 

Other published protocols use flash / isopentane frozen samples and nuclei isolation (e.g. Dronc-seq) if this is suitable for your tissue.

 

Hope this helps

Re: Fixed tissue dissociation and sequencing

Posted By: abaffyp, on Feb 8, 2018 at 2:45 AM

Hi Carl,

 

As first - we want to use RNAlater. From a lot of our experiments we know that RNAlater is not optimal for next steps. It causes problem with RNA isolation and RT reaction. It's optimal, only when you haven't another option (i.e. you need to send some samples over the world, and using of dry ice is not option). But I don't know anything about it's effect on direct tissue dissociation and next processing. But it's not so hard to test it, do you have protocol for direct dissociation of RNAlater fixed tissue?

 

Theoretically, PFA fixation is not problem for 3'sequencing. But, 10x use TSO, and probably TSO doesn't work correctly with degraded RNA.

 

I'm not familiar with nuclei isolation protocols, I need to check it, but I think it will not be usable for single-cell dissociation.

 

Thanks

 

Pavel

Re: Fixed tissue dissociation and sequencing

Posted By: rmtsoa, on May 7, 2018 at 11:03 AM

Hi Pavel:

 

I just read your response and have a question regarding the TSO reaction. Theoretically, TSO would bind to anywhere the reverse transcriptase falls off, did you check the length of the synthesized cDNA before fragmentation?

 

Thanks,

Roesmarie