Figure 1. Experimental design to measure the landscape of gene expression in the postmortem human locus coeruleus (LC) using spatially-resolved transcriptomics (SRT) and single-nucleus RNA-sequencing (snRNA-seq).

(A) Brainstem dissections at the level of the LC were conducted to collect tissue blocks from five neurotypical adult human brain donors. (B) Inclusion of the LC within the tissue sample block was validated using RNAscope (Maynard et al., 2020; Wang et al., 2012) for a pan-neuronal marker gene (SNAP25) and two norepinephrine (NE) neuron-specific marker genes (TH and SLC6A2). High-resolution hematoxylin and eosin (H&E) stained histology images were acquired prior to SRT and snRNA-seq assays (scale bars: 2 mm in H&E stained image; 20 μm in RNAscope images). (C) Prior to collecting tissue sections for SRT and snRNA-seq assays, tissue blocks were scored to enrich for the NE neuron-containing regions. For each sample, the LC region was manually annotated by visually identifying NE neurons in the H&E stained tissue sections. 100 μm tissue sections from three of the same donors were used for snRNA-seq assays, which included FANS-based neuronal enrichment prior to library preparation to enrich for neuronal populations. After all quality control (QC) steps, the final SRT and snRNA-seq datasets used for analyses consisted of samples from 4 and 3 donors, respectively.