Aggregated ATAC-Seq data for hepatocytes isolated from TA and NIC offspring (n = 4 animals each, with dexamethasone-treated and -untreated samples for each animal). (A) Multimegabase-scale differences in the accessibility landscape of TA and NIC hepatocytes. Top two panels show ATAC-Seq data for NIC and TA offspring for chromosome 6, along with an averaged NIC-TA score, followed by gene density. For chromosome 7, only NIC-TA and gene density are shown. Red boxes highlight a subset of genomic regions of low gene density in which TA hepatocytes exhibit greater ATAC-Seq signal than NIC hepatocytes. Conversely, gene-dense regions generally exhibit higher ATAC signal in NIC hepatocytes (not highlighted). The mechanistic basis for this global difference is unclear – it does not appear to reflect contamination of TA samples with dead cells, for example, as these samples (from nicotine-naïve animals) did not differ in viability, and plating of hepatocytes also effectively selects against dead cells. As fragment length distributions were consistent from library to library, it also seems unlikely that there were gross differences in the concentration or activity of the added Tn5. Nonetheless, while this difference could reflect meaningful biology, such as a global difference in heterochromatin condensation, global differences in any genome-wide assay should of course be viewed with skepticism. (B) ATAC-Seq data for 500 bp surrounding all annotated transcription start sites (TSSs), sorted from high to low average ATAC signal intensity. (C) Increased ATAC signal in NIC hepatocytes is shown for all TSSs, or RXRA or LXRB binding sites, as indicated. (D–H) Examples of loci exhibiting enhanced chromatin accessibility in NIC offspring, relative to TA offspring. (D–E) show ATAC-Seq tracks in which TA and NIC data are set to the same vertical range, as in Figure 4F. For panels F-H, y axes are set independently for TA and NIC datasets, visually correcting for the global differences between TA and NIC datasets. In these panels, a subset of significantly NIC-enriched peaks (Supplementary file 3) are indicated with arrows.