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

ABSTRACT: This submission contains processed single-nucleus RNA-seq feature-barcode matrices generated to validate fluorescence-activated nuclei sorting (FANS) strategies for enrichment of microglia from frozen human post-mortem brain. 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE333448 | GEO | 2026/07/24

REPOSITORIES: GEO

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Project description:IThe transcription factor (TF) interferon regulatory factor 8 (IRF8) controls both developmental and inflammatory stimulus-inducible genes in macrophages, but the mechanisms underlying these two different functions are largely unknown. One possibility is that these different roles are linked to the ability of IRF8 to bind alternative DNA sequences. We found that IRF8 is recruited to distinct sets of DNA consensus sequences before and after lipopolysaccharide (LPS) stimulation. In resting cells, IRF8 was mainly bound to composite sites together with the master regulator of myeloid development PU.1. Basal IRF8M-bM-^@M-^SPU.1 binding maintained the expression of a broad panel of genes essential for macrophage functions (such as microbial recognition and response to purines) and contributed to basal expression of many LPS-inducible genes. After LPS stimulation, increased expression of IRF8, other IRFs, and AP-1 family TFs enabled IRF8 binding to thousands of additional regions containing low-affinity multimerized IRF sites and composite IRFM-bM-^@M-^SAP-1 sites, which were not premarked by PU.1 and did not contribute to the basal IRF8 cistrome. While constitutively expressed IRF8-dependent genes contained only sites mediating basal IRF8/PU.1 recruitment, inducible IRF8-dependent genes contained variable combinations of constitutive and inducible sites. Overall, these data show at the genome scale how the same TF can be linked to constitutive and inducible gene regulation via distinct combinations of alternative DNA-binding sites. Bone marrow cells isolated from C57BL/6 or BXH2/TyJ mice were plated in 10 cm plates in 5ml of BM-medium (high glucose DMEM supplemented with 20% low endotoxin fetal bovine serum, 30% L929-conditioned medium, 1% glutamine, 1%, Pen/Strep, 0.5% Sodium Pyruvate, 0.1% M-NM-2-mercaptoethanol). Cultures were fed with 2.5 ml of fresh medium every two days. Macrophages were subjected to different treatment (see individual samples for details). Total RNA was extracted from 5x106 cells using RNAeasy kit (Qiagen). Libraries were prepared from 1-2 M-BM-5g of RNA, after oligo-dT selection, using the TruSeq RNA sample preparation kit (Illumina).

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

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