β agarase 1

Nucleic acids extracted from canine plaque samples were pooled and visualised in 1% low melting point agarose (Sigma-Aldrich) gels following electrophoresis. Nucleic acids corresponding to genomic DNA (≥ 20 Kb) and Small-SubUnit rRNA (16S and 18S, ca. 1 Kb) were excised from the agarose gel for purification.
Genomic DNA was purified from the agarose gel slice using the QiaQuick Gel Extraction kit (Qiagen) following the manufacturer’s protocol, and purified DNA was eluted into nuclease-free water and stored at -20°C until required. SSU rRNA was purified from agarose gels using β-Agarase I (New England Biolabs) following the manufacturer’s protocol with two modifications: 30 units of RNasin Plus Ribonuclease inhibitor (Promega) and 3 units of Turbo DNA-free (Ambion) were added. SSU rRNA was subsequently purified by precipitation with ¼ volume 10 M ammonium acetate and 2 x vol. 100% ice-cold ethanol and incubated at -80°C for 30 min. Following centrifugation at 18,000 g for 15 min, the RNA pellet was washed in 70% ethanol, resuspended in nuclease-free water and stored at -80°C until required.
Following purification, SSU rRNA and DNA from the four pools of five canine plaque samples were combined into a final pool of 20 samples prior to reverse transcription or 16S rRNA gene PCR, respectively.

Apoptosis of IMR90 and IMR91L cells was induced by exposing 2 × 10⁶ cells to either 1 μmol/L staurosporine (Cell Signaling Technology, Inc., Danvers, USA)/0.1% DMSO or 0.1% DMSO alone (as control) for four hours at 37°C. An aliquot of about 5-10 × 10⁶ cells/mL was co-stained with Annexin V-APC (BD Biosciences, San Jose, USA) and 7-Aminoactinomycin D (7-AAD, BD Biosciences, San Jose, USA) for 15 minutes to monitor the progress of apoptosis by FACS analysis.
The remaining cells were treated with lysis buffer (0.40 M Tris–HCl pH 8.0, 0.06 M Na-EDTA, 0.15 M NaCl, 1% SDS) and RNA was digested for 1 hour at 37°C using 15 μg/mL RNase A. 1 M sodium perchlorate and one volume chloroform were added to deproteinise cell lysates. DNA fragmentation was checked using the Genomic DNA Screentape on an Agilent 2200 Tap2station (Agilent, Santa Clara, USA) (see Additional file 9).
High molecular (>48 kb) and degraded apoptotic DNA (~4 kb) were extracted by cutting slices out of a preparative 1% low melt agarose gel and subsequent digestion with β-Agarase I according to the manufacturer´s protocol (New England Biolabs, Ipswich, USA).

DNA combing assay was carried out on the molecular combing system from Genomic Vision as described^{48 (link)}. Cells were pulse labeled sequentially with 50 μM CldU and 50 μM IdU (20 min each) and then incubated with 500 μM thymidine for 60 min. Cells were harvested and embedded in 0.75% low melting agarose plugs. Agarose plugs were melted at 70 °C for 20 min in 0.1 M MES (2-(N-morpholino) ethanesulfonic acid) (pH 6.5). The melted agarose was cooled down to 42 °C and incubated in the presence of β Agarase I (NEB, M0392) overnight. Silanized coverslips were dipped in the DNA solution and incubated for 2 min at RT and then combed and baked for 2 h at 60 °C to crosslink DNA to coverslips. Next, slides were denatured in 0.4 M NaOH for 20 min at RT, washed extensively with PBS and then stained using CldU and IdU antibodies, and single DNA molecules were stained by YOYO-1.