ExoChew-Seq: An in vitro method for investigating singlestranded DNA-protein interactions

During various cellular DNA transactions such as DNA replication and repair, genomic doublestranded DNA (dsDNA) is transiently unwound to generate single-stranded DNA (ssDNA). Several proteins interact with ssDNA, site- and/or structure-specifically, to regulate key cellular processes. Investigating these processes requires genome-wide identification of these ssDNA sequences. However, because of the transient nature of ssDNA in vivo techniques cannot reveal all such sequences requiring an in vitro alternative. Traditionally, ssDNA libraries are prepared by boiling or alkali-treatment of dsDNA libraries, but these techniques have their limitations as ssDNA can rapidly reanneal to generate dsDNA. We present here ExoChew, a novel enzymatic technique, which generates persistent ssDNA libraries from dsDNA libraries. In ExoChew, T7 exonuclease (T7 exo) or E. coli exonuclease III (Exo III), which binds and cleaves dsDNA in a specific direction, generates ssDNA libraries from dsDNA libraries. To validate the technique and demonstrate its applications, we conducted a genome-wide search for hnRNP K binding sites in an ExoChew-generated ssDNA library from mouse genomic DNA. The hnRNP K-bound ssDNA fragments were sequenced (ExoChew-seq) and a consensus hnRNP K binding motif was identified. Two other potential uses of ExoChew are the generation and genome-wide identification of ssDNA secondary structures and protein-ssDNA interactions. Overall design: Mouse cell line, NMuMG, and human cell lines, A549 and HeLa cells, were grown in DMEM with a high glucose medium containing 10% FBS. Cells were grown to sub-confluency in 10 cm plates, trypsinized, washed with PBS, and finally, lysed by adding 10 mM Tris (pH 7.4), 1 mM EDTA (pH 8), and 1% SDS. Chromosomal DNA was prepared using a Quick-DNA Midiprep Plus Kit. s. The Oxford Nanopore Ligation Sequencing Kit was then used to ligate adapters and prepare the dsDNA, according to the manufacturer's instructions. The dsDNA conversion products were then loaded and sequenced using an ONT MinION flow cell. MinKNOW software was used for controlling the device, data acquisition, and real-time basecalling using the manufacturer's recommended settings.