Hi,
I am isolating nuclei from snap frozen brain tissue (prefrontal cortex). The nuclei are then used to perform single-nuclei RNA seq using 10x genomics technology.
I normally extract the nuclei from the tissue and then proceed to GEM generation and cDNA synthesys in the same day, which is quite a long procedure.
I was wondering if anybody is freezeing the nuclei suspension before proceeding to the 10x application. If so, which freezing medium do you use (i know of BAM Banker medium for example)? do you loose a lot of nuclei in the freezing - thawing process?
Thank you in advance
Martina
Hi Martina,
We routinely freeze our isolated nuclei samples and bank them at -80C for later 10x snRNA-seq loading. We use FACS to sort particular populations and after FACS we spike in BSA into our sorted samples to bring the BSA concentration up to 2% (this prevents aggregation during centrifugation). We then centrifuge in a swinging bucket at 4C (600 xg for 5 minutes), remove the majority of the supernatant and resuspend the sample in freezing media. We use the following freezing media:
1 ml of Nuclei Freezing Media
730 µl Nuclease-free water
100 µl 10x PBS (1x final concentration)
100 µl DMSO (10% final concentration)
66.7 µL 30% BSA (2% final concentration)
3 µL RNAsin Plus RNAse inhibitor (0.3% final concentration)
Ahead of 10x loading, we thaw and centrifuge the samples to concentrate them for loading. During this process we typically lose ~25% of the nuclei. We tailor the number of nuclei per tube that we sort at FACS to compensate for this loss and we’ve found that sorting a minimum of 50,000 nuclei per tube allows us to hit ~700-1000 nuclei/ul after thawing and centrifuging.
-Rebecca