Single nuclei chromatin accessibility and transcriptomic map of breast tissues of women of diverse genetic ancestry [GeoMx]
Single nuclei analysis is allowing robust classification of cell types in an organ that helps to establish relationships between cell-type specific gene expression and chromatin accessibility status of gene regulatory regions. Using breast tissues of 92 healthy donors of various genetic ancestry, we have developed a comprehensive chromatin accessibility and gene expression atlas of human breast tissues. Integrated analysis revealed 10 distinct cell types in the healthy breast, which included three major epithelial cell subtypes (luminal-hormone sensing, luminal adaptive secretory precursor, and basal-myoepithelial cells), two endothelial subtypes, two adipocyte subtypes, fibroblasts, T-cells, and macrophages. By integrating gene expression signatures derived from epithelial cell subtypes with spatial transcriptomics, we identify specific gene expression differences between lobular and ductal epithelial cells and age-associated changes in epithelial cell gene expression patterns and signaling networks. Among various cell types, luminal adaptive secretory cells and fibroblasts showed genetic ancestry dependent variability as a subpopulation of luminal adaptive secretory cells with alveolar progenitor (AP)  cell state were enriched in Indigenous American (IA) ancestry and fibroblast populations were distinct in African ancestry. ESR1 expression pattern was distinctly different in tissues from IA compared to the rest, with a high level of ESR1 expression extending to AP cells and crosstalk between growth factors and Estrogen Receptor signaling is evident in these AP cells. In general, cell subtype-specific gene expression did not correlate with chromatin accessibility differences, suggesting that transcriptional regulation independent of chromatin accessibility governs cell type-specific gene expression in the breast. Overall design: FFPE sections from donors with two time tissue donations were selected for the study. Each slide contained two 5 micrometer sections from FFPE blocks. Each slide represented one donor with 2 barcodes representing 2-time donations. Each donor had three repeats (3 slides per donor). The sections were cut with Leica DB80 LS blades (Leica #14035843488) on a rotary microtome instrument (Leica RM2125 RTS) and placed on the center of a Superfrost Plus Microscope slide (Fisher scientific #1255015). Tissue sections were placed in the center of the slide and be no larger than 35.3 mm x 14.1 mm.  Regions of interest (ROIs) were selected after staining the slides with pan-keratin, alpha-SMA and FABP4 antibodies. All ROIs passed a sequencing quality control assessment. Next, negative control probes were used to estimate background and downstream gene detection and to remove outliers. The limit of quantification (LOQ) of each ROI was calculated using the geometric mean and geometric standard deviation of the negative control probes to identify genes detected above background in the experiment. All ROIs passed LOQ-based filtering with more than 1% of genes detected. Breast tissues from healthy donors donated 10 years apart; first number in the title column correspond to first donation and the second correspond to donation from the same donor 10 years later.