Alkaline Degradation and Sequencing Protocol

In total, 2 × 10⁷ cells were harvested by centrifugation and genomic DNA was prepared using Blood & Cell Culture DNA Midi Kit 100/G Genomic-tips (Qiagen #13343). To examine alkaline degradation 3 μg of DNA was treated with 0.3 M NaOH at 55 °C for 2 hr in 15 μl. The reaction was stopped by adding 3 μl of 1 M Tris-HCl (pH7.5). 1 μl of this solution was subjected to TapeStation RNA ScreenTape Analysis (Agilent #5067-5576, #5067-5577, #5067-5578) to detect ssDNA. Visualisation of DNA fragment patterns and quantification of DNA < 2.0 kb were performed using TapeStation Software (Agilent). For library preparation 25 μg of genomic DNA was alkali treated in 0.3 M NaOH at 55 °C for 2 h, then loaded onto a 1.5% agarose gel and run for 1 h 40 min at 100 V. The gel was stained with acridine orange (final concentration 5 μg/ml) for 2 h at room temperature with gentle shaking followed by overnight destaining in water. Fragments of 300–2000 bp were excised from the gel and isolated with a gel-extraction kit (Macherey-Nagel, NucleoSpin Gel and PCR Clean-up, #740609). Library preparation was performed as previously described^{5 (link),57 (link)}. Libraries were 150-bp paired-end (PE) sequenced on an Illumina Hiseq X platform (Macrogen, Tokyo, Japan).