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To examine the difference in sequence constraint within regulatorily active LINE transposable elements and avoid bias, we focused on those LINE elements that overlapped tissue-specific regulatory regions in all species. For Additional file 1: Figure S10D for each tissue-specific regulatory region associated with a LINE L1 or L2, we extracted the GERP rejected substation scores [98] from Ensembl version 98 [90]. Briefly, unalignable genomic regions do not have a GERP score, while a negative score in alignable indicates more sequence constraint than expected and a positive score indicates less. We plotted the resulting graphs in R version 3.6.2 [91] using ggplot2 version 3.1.1 [92] and calculated p values using the base R wilcox.test.

For Additional file 1: Figure S10E, we extracted the number of constrained elements, i.e., short genomic regions that have more sequence constraint than expected [98], for each tissue-specific regulatory region associated with a LINE L1 or L2 from Ensembl version 98 [90]. Unlike the analysis reported in Fig. Fig.5c,5c, this includes also unalignable genomic regions. We plotted the resulting graphs in R version 3.6.2 [91] using ggplot2 version 3.1.1 [92] and calculated p values using the base R chisq.test.

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