Adapted Smart-seq3xpress Facilitates Selective Microglial Transcriptomic Profiling From Frozen Brain Tissue

Area of Science:

• Neuroscience
• Immunology
• Genomics

Background:

• Microglia play a critical role in brain health and disease, particularly neuroinflammation.
• Single-cell RNA sequencing (scRNA-seq) is powerful for studying microglia but limited to fresh tissue.
• Analyzing microglia in cryopreserved or archival samples is challenging due to lack of specific protocols.

Purpose of the Study:

• To develop a method for microglial nuclei profiling from fresh-frozen brain tissue using Smart-seq3xpress.
• To enable high-resolution transcriptomic analysis of microglia in archival samples.
• To overcome limitations of standard single-nucleus RNA sequencing for microglial studies.

Main Methods:

• PU.1-based enrichment of microglial nuclei from fresh-frozen tissue after brief formaldehyde fixation.
• Thermolabile Proteinase K treatment to reverse cross-links for Smart-seq3xpress compatibility.
• Benchmarking in a mouse model of ischemic stroke.

Main Results:

• The developed method yielded higher gene and UMI counts and a greater proportion of coding reads compared to standard protocols.
• Transcriptomic profiles accurately reflected whole-cell RNA sequencing data.
• The method successfully detected microglial activation markers and subpopulations.

Conclusions:

• This approach enables high-resolution transcriptomic analysis of microglia from fresh-frozen and archival brain samples.
• It expands the utility of biobanked material for neuroinflammatory disease research.
• Facilitates basic and translational studies by providing cellular insights from preserved tissues.