Benzonase e1014

To investigate Top2a interaction partners, co-immunoprecipitation was performed from U2OS Nuclear Extract (15 mg/ml total protein). Top2a was immunoprecipitated using an anti-Top2a antibody (ab12318; Abcam) immobilized on Dynabeads Protein A magnetic beads (c/n 10001D; Thermo Fisher Scientific) according to the manufacturer’s instruction. Antibodies were cross-linked to beads using DPM (c/n 21666; Thermo Fisher Scientific) as recommended by the manufacturer. Beads were blocked with BSA in PBS overnight. 100 μl NE was diluted by dilution buffer (25 mM Tris HCl, pH 7.9, 12.5 mM MgCl₂, 10% glycerol, and 0.03% NP40) to a final KCl concentration of 150 mM and treated by 500 U of benzonase (E1014; Sigma-Aldrich) for 30 min at 4°C. 25 μl of the beads were added, and the suspension was incubated on a rotating wheel for 1 h at 4°C. Beads were washed three times with 100 μl wash buffer (25 mM Tris–HCl, pH 7.9, 150 mM KCl, 12.5 mM MgCl₂, 10% glycerol, and 0.03% NP40) and proteins were eluted by incubation in 1× LDS sample buffer (c/n NP0007; Thermo Fisher Scientific) at 65°C for 10 min. Immunoprecipitated proteins were analyzed by Western blot using anti-UBF, anti-RPA49, and anti-Top2a antibodies (sc-9131; Santa Cruz; 611413 BD Transduction; and ab12318; Abcam).

Porcine SIS was decellularized with an optimized protocol, which included mechanical agitation and the use of protease inhibitors, alternating with hypotonic and hypertonic solutions. We incubated tissue with a non-ionic detergent (Tergitol, 15S9, Sigma-Aldrich, Saint Louis, MO, USA) and an ionic one (Sodium Cholate, C1254, Sigma-Aldrich, Saint Louis, MO, USA) with intermediate washing cycles in PBS or saline solution, with a subsequential alcohol-based solution for tissue decontamination and delipidization, and endonuclease (Benzonase^®, E1014, Sigma-Aldrich, Saint Louis, MO, USA) treatment to remove the residual DNA fragments.
The resulting acellular tissue was treated with two different protocols (the first based on 70% ethanol/water and the second based on an antibiotic/antimycotic plus peracetic acid solution) to sterilize it in preparation for cytocompatibility tests.

Jejunum was collected from an abattoir and treated within 3 hrs. after animal sacrifice. The abattoir’s protocols followed EC guidelines 1099/2009 concerning animal health and protection at the time of sacrifice, was supervised by the Italian government, and was approved by the associated legal authorities of animal welfare (Food and Consumer Product Safety Authority). SIS isolation was obtained accordingly to an already described procedure [32 (link),40 (link),41 (link)] by removing the two external muscular layers and serosa and the internal tunica mucosa. Thereafter, SIS was rinsed in phosphate-buffered saline (PBS) and decellularized following the optimized procedure described in [32 (link)]. Decellularization is based on the use of protease inhibitors, hypo- and hyper-tonic solutions, Tergitol 15S9 (Sigma-Aldrich, Saint Louis, MO, USA) and Sodium Cholate C1254 (Sigma-Aldrich, Saint Louis, MO, USA), with a subsequent alcohol-based solution. Finally, an endonuclease (Benzonase^®, E1014, Sigma-Aldrich, Saint Louis, MO, USA) was used to remove DNA residues.