Decomposition of adipose tissue into subtypes has been challenging because the fragile nature of adipocyte. This protocol was developed to overcome this by effective isolation of single nuclei. This workflow provides highly purified single nuclei with minimized nuclear aggregates and cellular debris.
This is advantageous to single-cell transcriptome profiling of thousands of cells. This simple and robust protocol can be applied to study tissue-level organization of adipocyte in their adipose resident cells. And in particular, to development phenotyping, adipose knockout and transgenic mice.
Visual demonstration of this protocol is critical so that people who don't have much experience in handling adipocyte can replicate and insight into adipose heterogeneity. After collecting adipose tissue from mice, pat it dry with a paper towel, and place it in a clean dish. Add calcium chloride to the digestion buffer to a final concentration of 10 mM.
Then, add a small volume to the tissue and thoroughly mince it. Add about half the amount of prepared digestion buffer to the minced tissue. And use a serological pipette to transfer the sample to a 50 mL centrifuge tube.
Wash the dish with the remaining buffer and add it to the 50 mL tube. Pipette the sample up and down several times. Then, incubate it for 12 to 15 minutes at 37 degrees Celsius while shaking at 200 to 210 rpm.
After the incubation, add 0.5%BSA to the sample at a 1 to 1 volume ratio and mix well by pipetting. Centrifuge the sample at 300 xg for five minutes at room temperature to spin down the stromovascular fraction leaving the adipocyte fraction on top. Filter the top fraction though a 400 m cell filter into a 50 mL tube.
Then, flip the filter upside down over the tube and transfer the adipocytes to a a new tube using 0.5%BSA to reverse-wash the filter. Bring the total volume of the BSA to 10 to 15 mL and pipette the sample up and down with serological pipette. Then, centrifuge it again at 300 xg for five minutes at room temperature.
After centrifugation, use a wide bore pipette tip to carefully transfer the top layer of the sample to a 100 m cell filter on top of a new pre-chilled tube on ice. Rinse the filter with a sufficient amount of nuclei preparation buffer, and then discard it. From this step forward, keep a sample on ice and work quickly.
Leave the sample on ice for up to two minutes intermittently inverting the tube to mix. Then, centrifuge at 1000 xg and 4 degrees Celsius for 10 minutes. Remove the supernatant and resuspend the pellet in 1 mL of nuclei preparation buffer.
Transfer the sample to a clean microcentrifuge tube taking care of to avoid touching the walls of the old tube. Add 0.6 units per L RNase inhibitor and mix it. Then, centrifuge the sample for another 10 minutes.
Remove the supernatant and resuspend a pellet and 1 mL of nuclei wash buffer. Double filter the sample into a clean tube using stackable 30 m filters. Then, wash the filters with about 300 L of nuclei wash buffer.
To confirm successful isolation of healthy nuclei, stain a small aliquot of the sample with trypan and use an automated cell counter to assess average dead size and percent of dead cells. Stain in a small aliquot of the sample with DAPI and examine it under a microscope with a 20X objective or higher. The nuclei should be round and have an intact nuclear membrane.
White arrows indicate nuclei without an intact membrane. After cleaning up the nuclei with FACS, concentrate this sample by centrifuging at 500 xg for five minutes at 4 degrees Celsius. Then, reconstitute it in an appropriate volume of nuclei wash buffer.
To achieve a 50 to 1500 nuclei per L concentration and proceed with single-cell nuclei RNA seq. To remove cellular debris and doublets, adipocyte nuclei are sorted for size and granularity to exclude nuclear aggregates and multiplets. And finally for DAPI positive events.
This grading strategy result in highly purified single-nuclei with minimal aggregates and debris. When inspected by microscopy, the sorted nuclei should be round and have an intact membrane. Successful purification of the nuclei can also be performed with real-time qRT-PCR using primers for nascent Ucp1 mRNA, a marker gene for brown adipocytes.
The chromium platform determined a transcriptome of 7500 nuclei from murine interscapular brown adipose tissue. Seven different cell types were revealed with t-SNE dimensionality reduction and k-means clustering. All clusters expressed Fabp4, a pan adipocyte gene and a subset expressed a high level of Ucp1 mRNA.
Nuclei isolated and then confirmed though this protocol can be subjected to virtually any single-cell level gene expression platform including Chromium from 10x Genomics.
