Human genomic DNA is widely interspersed with i-motif structures

DNA i-motif structures are formed in the nuclei of human cells and are believed to provide critical genomic regulation. While the existence of i-motif structures in human cells has been demonstrated by immunofluorescent staining and by the characterisation of select model genes, the abundance and distribution of such structures in the human genome have remained unclear. Here we utilise high affinity i-motif immunoprecipitation followed by sequencing to map i-motifs in the genomic DNA of human MCF7, U2OS and HEK293T cells. Validated by biolayer interferometry and circular dichroism spectroscopy, our approach identified i-motif structures that are widely distributed throughout the human genome and are common in genes upregulated in G0/G1 cell cycle phases. Overall design: MCF7, HEK293T and U2OS cells were grown, harvested and pelleted following conventional protocols. Protein and RNA depleted genomic DNA was extracted, purified and fragmented by sonication. Antibody incubation was perfomed at 4 °C using an i-motif specific antibody (Biotinylated iMab scFv) to pull-down putative i-Motif forming sequences followed by sequencing using illumina NovaSeq 6000 and NovaSeq 6000 SP reagent kit in a paired-end 300 cycles mode following the manufacturer protocols . Data was finally collected from two biological replicates for each cell line and sequenced input DNA from each replicate was used for peak calling analysis. Additinally, DNA derived from MCF7 cells by a modified protocol incubating purified DNA and the iMab antibody at 16 °C was also sequenced to inviestigate temperature bias.