(A) Schematic outline of the isolation, purification, and culture of brain ECs. (B) Scatter plots comparing cross-sample normalized RNA-seq read counts for all protein-coding genes (gray symbols) between acutely isolated adult brain ECs (y-axis; average of two independent samples) and primary brain ECs cultured for 8 days (x-axis; average of six independent samples). The left plot highlights (in red) EC-enriched genes that are expressed at similar levels in adult brain, P7 brain, P7 liver, P7 lung, and P7 kidney ECs. The right plot highlights (in blue) genes associated with the BBB (i.e., that are >2 fold enriched in brain ECs relative to liver, lung, and kidney ECs). The majority of BBB genes show reduced expression in culture. (C) Heatmap depicting log2 transformed transcript abundances from cultured brain ECs, and from acutely isolated P7 and adult brain ECs, and P7 liver, lung, and kidney ECs for 993 tissue-specific EC genes. (D) Principal component analysis (PCA) based on the abundances of all protein-coding transcripts (top), EC-enriched transcripts (center), and pan-EC transcripts (i.e. transcripts common to adult brain, P7 brain, P7 liver, P7 lung, and P7 kidney ECs; bottom). The symbols for each sample represent biological replicates. Arrows indicate cultured EC data points. In the PCA in the bottom panel, the two samples in the upper right were replicates isolated by Method B, and their RNA-seq libraries were generated and sequenced by a different facility compared to the other four samples, which were isolated by method A (see Materials and methods). (E) Transcript abundances for six genes from each of the four indicated categories of transcripts. Labels at the bottom of the figure apply to all panels above.