14Gene expression requires specific structural alternations in the nucleoid structure to enable the access 15 of the transcription machinery into the genomic DNA. In prokaryotes, DNA binding proteins, 16including nucleoid-associated proteins (NAPs) and transcription factors (TFs), drive the change in 17 structure and gene expression. Currently, studies of global NAP and TF binding are often hindered by 18 the lack of appropriate epigenomic tools. Here, we present POP-seq, a method that provides in vivo 19 genome-wide openness profiles of the bacterial nucleoid. We demonstrate that POP-seq can be used 20 to map the global in vivo protein-DNA binding events. Our results highlight a negative correlation 21 between genome openness, compaction and transcription, suggesting that regions that are not 22 Genome organization is crucial to all life forms. In eukaryotes, histone oligomers organize the 35 chromosomal DNA into nucleosomes of defined sizes, the building blocks of higher-order structures. 36By contrast, such well-defined structures are lacking in bacteria. Instead, a wide variety of poorly 37 conserved nucleoid-associated proteins (NAPs) control the dynamic organization of the nucleoid and 38 directly affect how genetic information is accessed, interpreted, and implemented 1,2,3,4 . Among the 39 most widely studied NAPs is H-NS in E. coli 5 and its functional analog, Rok in B. subtilis 6 , both 40 known to have high affinity towards AT-rich regions 7,8 . 41Omic technologies have revolutionized molecular biology by providing accurate measurements of 42 molecular components, such as protein, RNA, and cis-acting elements. Yet, there currently exist few 43 techniques for comprehensive identification and assessment of dynamic NAP binding and nucleoid 44 organization in vivo. Implementation of HiC and similar methods have provided vital insights into the 45 3 three-dimensional structure of the chromosome. However, HiC is limited by its technical and 46 bioinformatic intricacy, the need for extreme sequencing depth, and the prerequisite for highly 47 16 ). Further, we found that these signals are more prominent over transcription start sites (TSSs) ( Fig. 127 2e), which is the center of transcriptional regulation. Our results imply that the nucleoid is more open 128 at promoter sites, consistent with what is described in eukaryotes 22 , and that the signals could be 129 originating from transcription factor-DNA binding events. 130Thus, we examined the Tn5 tagmentation sites (POP-seq footprints), in which we only scored the 9 131 nucleotides covered by the Tn5 transposase for each aligned read. As a result, we generated two 132 genome-wide alignment files for each experiment: one for the forward-strand sequencing reads and 133 another for the reverse-strand sequencing reads. We tested if local depletions (footprints) in the two 134
