To perform motif footprinting, we first identified the coordinates of each instance of the motif to be footprinted using the matchMotifs function from the motifmatchr package with out = ‘positions’ to return the genomic coordinates of each motif instance (https://bioconductor.org/packages/motifmatchr). Motif coordinates were then resized to include the ±250-bp sequence. The Tn5 insertion frequency was counted at each position in the region for each motif instance to produce a matrix containing the total observed Tn5 insertion events at each position relative to the motif center for each cell. We then found the expected Tn5 insertion frequency matrix by computing the hexamer frequency matrix, M. The hexamer frequency matrix M was defined as a matrix with i rows corresponding to i different DNA hexamers and j columns corresponding to j positions centered on the motif and each entry Mij corresponded to the hexamer count for hexamer i at position j. To find the expected Tn5 insertion frequency at each position relative to the motif given the Tn5 insertion bias (see above), we computed the matrix cross product between the hexamer frequency
