Figure 2.

Effects of linker length on the integration reaction by Tn5 transposase. (a) Schematic of the dinucleosome substrates. (b) The dinucleosomes containing the 0-, 5-, 10-, 15-, 20-, 25- or 30-base-pair linker DNAs (containing 0.01 µg µl⁻¹ DNA) were incubated with the Tn5–DNA complex (0.5 µM). After deproteinization, the DNA fragments were analysed by non-denaturing PAGE with SYBR Gold staining. The bands marked with * are annealing products of the DNA fragments containing partially single-stranded DNA. Black, white and grey arrowheads indicate longer, middle and shorter DNA fragments produced by the Tn5 transposase reaction, respectively. The substrate DNAs exhibited unusual migration profiles, which do not correspond to the DNA length, probably due to the structural nature of the 601 sequence. The DNA sequences of these template DNAs were confirmed by direct sequencing. (c) Profile of the Tn5 transposase cleavage sites for the dinucleosome containing the 30-base-pair linker DNA. The DNA fragments tagged by the Tn5 transposase assay were analysed by massively parallel paired-end sequencing, and the 5'-ends of the fragments are mapped on the substrate DNA sequence, as described in the Methods. The two major cleavage sites are shown in the schematic.