May 2, 2022 / Neuroscience

Top tips for better neural sample prep

Josh Azevedo

The central nervous system (CNS) is many things—chief among them is "complex''. Looking at the facts, it makes sense: there are more cells in our brains (~170 billion) than there are stars in our galaxy, and each cell can have up to 10,000 connections. The type, transcriptomic program, and spatial arrangement of each of these cells can be unique—and offer key insights into CNS function in health and disease.

This biological complexity translates to technical challenges when working with neural samples: how do you choose between cells and nuclei for your starting sample? What do you need to do to get clean samples for single cell sequencing? How can you optimize tissue for spatial analyses? We put together these highlights from our Top 10 Neural Sample Prep Tips guide to help you answer these questions (and more).

Choosing the right fit—single cells or nuclei?

The choice of using cells or nuclei for your single cell work is a critical decision that will be tailored to the needs of your specific experiments.

  • What tissue types do you have available? You might have the freedom to choose whichever type of tissue you wish, or you may be limited to specific tissue types. Both fresh and fixed tissues will allow you to choose between extracting single cells or nuclei; however, frozen tissues will require the use of nuclei.
  • What analytes will you be studying? The specific assay you choose might have input requirements: single cell GEX, for instance, is compatible with both cells and nuclei. ATAC, however, requires nuclei as input, while V(D)J and cell surface profiling require whole cells.   
  • What cell types are you interested in? After experimental design considerations come technical considerations. Some larger CNS cell types, such as neurons, are difficult to dissociate from tissue and require the use of nuclei. Cells with low RNA content (e.g., microglia) may be difficult to resolve, so nuclei would be favorable.

Optimization makes all the difference

Optimizing your neural sample prep helps your lab ensure reproducibility, obtain reliable data, and save both time and resources. However, the cellular and anatomical heterogeneity of neural tissues means you’ll need to tailor your sample prep to your sample type, experimental approach, and to whether you’re looking at single cell or spatial transcriptomics.

Myelin can represent a major hurdle for single cell preps from the brain as it causes cells and nuclei to clump together, contributing to the debris in "dirty" preps and clogging microfluidics devices. While the myelin content in starting tissues varies depending on brain region and developmental stage, building myelin cleanup strategies into your samples—as well as QC checks of your final pre–library prep suspension—is critical.

Spatial approaches, meanwhile, can require fine tuning. Your optimal sectioning temperature, thickness, and permeabilization time will depend on your neural tissue type (brain versus eye versus spinal cord), fixation status, and disease type. Harder tissues (e.g., solid tumors) may require different sectioning conditions, while extremely soft tissues (e.g., eye and embryonic brain) should be simultaneously frozen and embedded to preserve morphology.

Getting more tips

Single cell and spatial tools are helping neuroscientists reshape the way we understand the CNS, and behind each experiment is a sample prep. Incorporating new tips can help you improve your neural sample processing workflows, so come check out the rest of our Top 10 Tips for preparing neural samples!