A roadmap for enriching rare cell populations from human post-mortem brain, demonstrated by 100% microglial purity (scRNA-Seq)

Nuclei were isolated from neurologically unaffected donor tissue from parietal cortex, occipital cortex, and cerebellum, then sorted using panels based on NeuN, OLIG2, IRF8, PU.1, and SOX9. The study benchmarks single-marker and multi-marker nuclear sorting strategies, including IRF8-positive enrichment, PU.1-positive enrichment, SOX9-positive astrocyte sorting, SOX9 dump-channel exclusion, and an extended four-channel panel enabling parallel recovery of neurons, oligodendrocytes, microglia, astrocytes, and an all-negative population.

Initial validation experiments used 10x Genomics Chromium Single Cell 3' v3.1 chemistry. Subsequent validation used Illumina Single Cell 3' RNA Prep, T2, based on PIP-seq chemistry, as a lower-cost orthogonal workflow for routine sort-quality assessment.