Total microbial genomic DNA, including bacterial and fungal genomic DNA in the feces of piglets, was extracted using a combined method of cetyl trimethyl ammonium bromide (CTAB) and bead-beating. Briefly, 0.25–0.30 g frozen feces were re-suspended in 1.5 ml ice-cold PBS and then were centrifuged at 9000 rpm for 10 min at 4°C to obtain microbial pellets. The pellets were washed in ice-cold PBS repeatedly until the supernatant became clear. Subsequently, the microbial pellets were re-suspended in 800 μl CTAB buffer containing 50 mM CTAB, 1.4 M NaCl, 100 mM Tris-HCl, 20 mM Ethylene Diamine Tetraacetic Acid (EDTA) and then were lysed by beat-beading using FastPrep-24 bead beater (MP Bio) at the top speed for total 240 s with an ice-cold bath for 120 s at the interval. After incubation at 70°C for 20 min, homogenate solution was centrifuged at 10,000 rpm for 10 min to obtain the supernatant. Five microliter of RNAase (10 mg/ml) was added into the supernatant obtained and the solution was incubated at 37°C for 30 min to remove the RNA. After that, three rounds of phenol: chloroform: isoamyl alcohol (V/V/V = 25: 24: 1) extraction were performed. The microbial DNA obtained was precipitated with the solution containing 1.5 ml ice-cold 95% ethanol and 40 μl 3M NaAc (20:1) at −20°C overnight and then re-suspended in 50 ml of Tris-EDTA buffer. Microbial genomic DNA was quantified using Qubit® 3.0 Fluorometer (Life technology) and DNA integrity was determined by gel electrophoresis (concentration of agarose gel: 1%, voltage: 150 V, and electrophoresis time: 40 min). Finally, the DNA samples examined were stored at −80°C until processing.
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