We performed transcription factor motif footprinting following previously described methods50. To account for Tn5 sequence insertion bias, we first computed the observed Tn5 insertion frequency at each DNA hexamer using all Tn5 insertions on chromosome 1. This was performed by extracting the base-resolution Tn5 insertion positions for each fragment mapped to chromosome 1 and extending the insertion coordinate 3 bp upstream and 2 bp downstream. We then extracted the DNA sequence corresponding to these coordinates using the getSeq function from the Biostrings R package (https://bioconductor.org/packages/Biostrings) and counted the frequency of each hexamer using the table function in R. We next computed the expected Tn5 hexamer insertion frequencies based on the frequency of each hexamer on chromosome 1. We counted the frequency of each hexamer using the oligonucleotideFrequency function in the Biostrings package with width = 6 and names = ‘chr1’, using the hg38 genome via the BSgenome R package (https://bioconductor.org/packages/BSgenome). Finally, we computed the Tn5 insertion bias as the observed Tn5 insertions divided by the expected insertions at each hexamer. This was performed using the InsertionBias function in Signac.
