Optimized Bile DNA Extraction Protocol

Total DNA extraction from 1-ml bile samples was performed following an optimized protocol based on a previously described method [19 (link)], with slight modifications. Briefly, samples were centrifuged at maximum speed at room temperature for 10 min and pellets re-suspended in 0.5 ml of extraction buffer consisting of 200 mM Tris-HCl pH 7.0, 25 mM EDTA, 250 mM NaCl, and the following enzymes: 20 mg/ml lysozyme (Merck, Darmstadt, Germany), 5 μg/ml of lysostaphin (Sigma-Aldrich, Saint Louis, MO, USA), and 40 U/ml mutanolysin (Sigma-Aldrich). Enzymatic lysis was performed for 1 h at 37 °C; after that, SDS was added to a final concentration of 0.5% (w/v), and mechanical disruption was performed in a FastPrep FP120 apparatus (Qbiogene, Carlsbad, CA, USA). The lysate solution was treated with proteinase K, 1.5 M NaCl as previously described [39 (link)] and extracted with phenol/chloroform. Precipitation of DNA was performed with 0.1 volumes of 3 M sodium acetate pH 5.2 and 2.5 volumes of cold ethanol. The DNA was then pelleted, washed with 70% ethanol, resuspended in 50 μl of molecular-biology grade water (Sigma-Aldrich), and stored at − 20 °C until use. DNA concentration and quality was determined in a BioTek Epoch™ spectrophotometer system (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and in parallel in a Qubit fluorometer with dsDNA assay kits (Thermo Fisher Scientific).