Collectively invading and single cells exhibit distinct transcriptional and epigenetic programs. (A) Heat maps from RNA sequencing data for each pair-wise comparison. Scale denotes z scores from log₂-normalized expression counts of most DEGs. (B) Principal component (PC) analysis plot of leaders, followers, and singles based on RNA sequencing data (n = 3). (C) Volcano plots denoting DEGs for each pair-wise comparison. DEGs (small, unbordered red dots) were classified as gene transcripts with −Log₁₀ P values of >1.3 (y-axis) and a log₂ fold change difference of >2.0 or less than −2.0 (x-axis). Bordered red dots are select genes overexpressed in collectively invading cells and bordered green dots are select genes overexpressed in single cells. (D) Number of DEGs between each pair-wise comparison. (E) RNA counts for Cdh1, Cldn4, Tacstd2, Esrp1, Lama3, and Krt14 in leaders, followers, and singles (n = 3). (F) Heat map of mouse methylation beta values between leaders and singles and representation of the percentage of hypermethylated regions across all DMRs between leaders and singles. Scale on heat map denotes beta value difference values for each DMR. L-1, L-2, and L-3 denote three replicates of leaders and S-1, S-2, and S-3 denote three replicates of singles. (G) Annotation of DMRs in singles when compared to leaders. (H) Beta value comparison of CpG loci within the promoter region of Cdh1 (n = 7), Cldn4 (n = 4), Tacstd2 (n = 5), Esrp1 (n = 6), Lama3 (n = 7), and Krt14 (n = 3) between leaders and singles. For all panels: mean ± SEM is shown. Unless noted, n.s., no significance, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.