Question: Are there any recommendations for post-mortem brain nuclei?
Answer: For a successful nuclei experiment, it is essential to have a clean prep of intact nuclei, free of debris and aggregates. A good nuclei prep results in accurate counts and high UMI diversity.
A customer-developed protocol for isolating nuclei from frozen tissue is available on our community website. This protocol was developed using post-mortem brain.
Steps for optimization of this protocol include:
- Addition of a cleanup step using Optiprep and/or a modified sucrose gradient if lots of debris and/or myelin remain in the suspension. The Optiprep gradient is very efficient at cleaning debris and myelin from adult brain tissue in a single step.
- Increasing the BSA concentration in the sample buffer to 2% if nuclei aggregation occurs.
- Assessing nuclei isolation by evaluating the cell lysis efficiency under the microscope. The cellular membrane should be solubilized while the nuclear membrane should be seen intact. Trypan blue will stain lysed cells and nuclei.
Products: Single Cell Gene Expression, Single Cell Immune Profiling