Fig. 3

Using biotinylated adapters complicates the DNAse Hi-C protocol and results in undesired by-products. a Schematic description of possible ligation reaction products obtained in the presence of biotinylated adapters from Ma et al. [22]. The adapters used by Ma et al. are referred to as bridge and blunt oligonucleotides. As evident from the scheme, sequence similarity between bridge and blunt adapters leads to the formation of multimers. b Ligation assays showing the formation of adapter multimers. Lanes 1 and 10–100-bp ladder. Lanes 2 and 3—bridge (2) and blunt (3) oligonucleotides. Lanes 4–6—self-ligation assays performed with bridge (4), blunt (5), and bridge/blunt oligonucleotides mix (6). Lanes 7–9—bridge (7), blunt (8), and bridge/blunt oligonucleotides mix (9) subjected to ligation, followed by phosphorylation and an additional round of ligation, which imitated enzymatic steps during the DNAse Hi-C protocol. The arrow shows the adapter multimerization products. Lane 11—self-ligation assay of the redesigned blunt adapter, which lacks sequence similarity with the bridge adapter. Lane 12—redesigned blunt adapter subjected to ligation, phosphorylation and an additional round of ligation. Lanes 13 and 14—the same reactions as in lanes 11 and 12 performed using a mix of bridge and redesigned blunt-adapter oligonucleotides