Hi all! Wondering if anyone is familiar with a Cre-inducible transgenic line where the nucleus of neurons could be marked with a reporter (GFP, Tdtomato, not picky…). Going to try single-nuc on a subpopulation of cells
Hi @ShanTan. @achamess has uses this strategy previously: Transcriptional Profiling of Somatostatin Interneurons in the Spinal Dorsal Horn | Scientific Reports.
Wow, so helpful. Thank you for sharing @thicunha. @achamess, hoping to ask you a couple of questions about your protocol. Mostly, did you find there was lots of debris (i.e. fat cells) when sorting your nuclei? I don’t see any gradient steps - does the fat separate from other cells when forming a pellet? Thanks!
No gradient. Just FACS. It cleans up the nuclei better than anything else, including gradients.
Also, use the Sun1-GFP mouse. It’s excellent. 021039 - CAG-Sun1/sfGFP Strain Details
Thanks very much @achamess. The mice are ordered! Did you find there was lots of debris when you brought your sample for sorting?
Yes. Lots of debris. That’s why FACS. If you just try to run that through 10x or whatever, it’ll get all that gunk and diminish the quality of your results.
I have a detailed protocol here