Single-cell sequencing of the human midbrain reveals glial activation and a neuronal state specific to Parkinson's disease

Data analysis workflow to reproduce the findings of the manuscript: "Single-cell sequencing of the human midbrain reveals glial activation and a neuronal state-specific to Parkinson's disease". Raw-data is publically available in the Gene Expression Omnibus (GEO) with the accession number GSE157783.

General data analysis workflow

Single-cell RNA sequencing

We investigated 11 human midbrain sections using the 10X scRNAseq solution: 5 IPD patients and 6 sex- and age-matched controls. The CellRanger 3.0 pipeline was used to count the transcripts. Scrublet was used to identify and filter out dublets. Seurat3 and monocle3 R (version > 4.0) packages were used for data normalization, sample integration, clustering, and trajectory inference. Individual functions for each of these steps are called from bash workflow scripts.

Reads mapping and UMI count

UMI count matrix and cell metadata are available as supplementary files in the GEO accession number GSE157783. Reads were aligned on the 10X pre-indexed reference human genome (hg19, GRCh38).

Preprocessing, duplet-scoring, cell-filtering, sample normalization & integration, clustering & cell-type annotation

./scrnaseq/run_analysis_midbrain.sh

Image analysis

We used fluorescence microscopy to validate the single-cell sequencing results. We analyzed microscopy images of two whole-midbrain ventral sections. The tissues were stained for neuronal, dopaminergic, and glial cells (oligodendrocytes, astrocytes, and microglia) markers, as well as for the Cadps2 protein.

Genetic enrichment