Single-Cell RNA Sequencing Analysis of Microglial Heterogeneity Protocol
Phase 1: Microglial Isolation from Human Brain Tissue (Days 1-10)
Tissue Acquisition: Obtain human brain tissue from post-mortem donors (n=8, ages 60-85) with Alzheimer's disease (AD, Braak stage IV-VI, n=4) or age-matched neurologically normal controls (n=4). Process tissue within 6 hours of death (PMI < 6 hours). Dissect hippocampal CA1 region (200-300 mg) from frozen blocks.
Microglial Enrichment: Dissociate tissue using Neural Tissue Dissociation Kit (Miltenyi #130-093-468) per manufacturer protocol. Pass through 70 μm cell strainer. Label CD11b+ cells with magnetic microbeads (Miltenyi #130-093-636) and enrich via MACS LS columns. Verify purity via flow cytometry (CD45+CD11b+ > 85% threshold).
...Single-Cell RNA Sequencing Analysis of Microglial Heterogeneity Protocol
Phase 1: Microglial Isolation from Human Brain Tissue (Days 1-10)
Tissue Acquisition: Obtain human brain tissue from post-mortem donors (n=8, ages 60-85) with Alzheimer's disease (AD, Braak stage IV-VI, n=4) or age-matched neurologically normal controls (n=4). Process tissue within 6 hours of death (PMI < 6 hours). Dissect hippocampal CA1 region (200-300 mg) from frozen blocks.
Microglial Enrichment: Dissociate tissue using Neural Tissue Dissociation Kit (Miltenyi #130-093-468) per manufacturer protocol. Pass through 70 μm cell strainer. Label CD11b+ cells with magnetic microbeads (Miltenyi #130-093-636) and enrich via MACS LS columns. Verify purity via flow cytometry (CD45+CD11b+ > 85% threshold).
viably: Assess cell viability via Trypan blue (目标 > 80% viable). Exclude samples with viability < 70%.
Phase 2: scRNA-seq Library Preparation (Days 11-17)
Single-Cell Capture: Load 10,000 cells per channel on 10x Chromium Controller (v3.1 chemistry, 5' kit). Target 5,000-8,000 captured cells per sample. Run RT reaction (1 hour, 72°C), cDNA amplification (12 cycles), and library construction per 10x protocol.
Sequencing: Pool libraries at equimolar concentrations (150 pM). Sequence on Illumina NovaSeq X Plus (150 bp paired-end, minimum 50,000 reads/cell). Target sequencing depth: 30,000 mean reads/cell.
Base Call and demultiplexing: Generate FASTQ files via Illumina bcl2fastq2. Demultiplex via cellranger mkfastq. Process raw data through cellranger count (GRCh38 reference, 10x标配).
Quality Control: Filter cells using Scrublet (doublet detection, expected doublet rate 5-8%) andempty drops (ambient RNA removal, FDR < 0.01). Remove cells with >5% mitochondrial reads or <200 detected genes. Retain cells with 500-5,000 genes detected.
Clustering and Annotation: Normalize counts via SCTransform. Run PCA (top 30 PCs),UMAP (n_neighbors=15, min_dist=0.3), and graph-based clustering (Louvain algorithm, resolution=0.4-1.0). Annotate clusters using known microglial markers: homeostatic (P2RY12, TMEM119, CX3CR1), disease-associated microglia (DAM, TREM2, APOE, ITAGAX), andpro-inflammatory (IL1B, TNF, CCL2).
Differential Expression and Trajectory Analysis: Compare cluster composition between AD and control using Wilcoxon rank-sum test (Bonferroni-corrected p < 0.05). Run pseudotime analysis via Monocle3 or Palantir to infer trajectory relationships among microglial states.