Journal: eLife

Article Title: Control of innate olfactory valence by segregated cortical amygdala circuits

doi: 10.7554/eLife.104677

Figure Lengend Snippet: Related to . ( A ) Representative images of tissue microdissection sites from aplCoA and pplCoA following extraction and DAPI staining (blue). Scale bars, 500 µm. ( B ) Location of all tissue sample sites used for snRNA-seq, color coded by plCoA zone (n=3 pools per zone, 4–11 sections per pool). Scale bars, 500 µm. ( C ) Validation of nuclear enrichment after FANS. Ethidium homodimer-1 (EthD-1, red) labels nuclei on a hemocytometer after sorting, with an absence of non-nuclear, EthD-1-negative debris. Scale bar, 100 µm. ( D ) Common gating strategy for FANS sorts for snRNA-seq in a representative sample. Far left, morphology gate on forward and side scatter area excludes likely debris. Middle left, forward scatter gate excludes nuclear doublets with high forward scatter width. Middle right, side scatter gate excludes nuclear multiplets with high side scatter width. Far right, stoichiometric DRAQ7+ fluorescence allows enrichment of single nuclei and exclusion of debris and multiplets. ( E ) Absolute number and proportion of snRNA-seq nuclei passing quality control filters from each replicate in each plCoA zone (n=27,726 in aplCoA, 19,406 in pplCoA, 3 libraries/batches each). ( F ) Violin plot of UMIs detected per snRNA-seq nucleus for each replicate, filtered at the median per library +five times the median absolute deviation within each library (median 6081 UMIs/nucleus). ( G ) Violin plot of genes detected per nucleus from each replicate, filtered at a minimum of 1000 features per nucleus (median 2547 genes/nucleus). ( H ) Percent mitochondrial gene UMIs per snRNA-seq nucleus, filtered at median +five times the median absolute deviation per library (median 0.02% mitochondrial UMIs/nucleus). ( I ) Percent ribosomal gene UMIs per snRNA-seq nucleus, filtered at median +five times the median absolute deviation per library (median 0.17% ribosomal UMIs/nucleus). ( J ) Principal component analysis of pseudobulk snRNA-seq samples created from each batch, colored based on their combination of zone and batch identity. ( K ) Evaluation of transcriptomic homology between batches, where the distance matrix is based on Spearman correlation between median expression of highly variable features for the whole dataset, and the dendrogram was created via hierarchical clustering of batches on this correlation matrix. ( L ) UMAP of all snRNA-seq nuclei colored by both target region and batch identity. ( M ) Relative proportion of nuclei of each type for all snRNA-seq batches. Brain diagrams were reproduced from .

Article Snippet: Nuclei were finally resuspended in nuclear flow buffer containing 3 μm DRAQ7 (Cell Signaling Technology) and again filtered through a 40 μm Flowmi cell strainer into a 5 ml round-bottom polystyrene tube.

Techniques: Laser Capture Microdissection, Extraction, Staining, Biomarker Discovery, Fluorescence, Control, Expressing