Phenol chloroform isoamyl alcohol

Total bacterial DNA was extracted as described previously [6 (link)]. Briefly, the fluid samples were incubated with 750 μg/mL lysozyme (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 90 min. Then, 10 μL purified achromopeptidase (Wako Pure Chemical Industries, Ltd., Osaka, Japan) was added at a concentration of 10,000 U/mL and incubated at 37 °C for 30 min. The suspension was treated with 60 μL 1% sodium dodecyl sulfate and 1 mg/mL proteinase K (Merck Japan, Tokyo, Japan), and incubated at 55 °C for 5 min. The lysate was treated with phenol/chloroform/isoamyl alcohol (Wako Pure Chemical Industries, Ltd.) and chloroform (Life Technologies Japan, Ltd., Tokyo, Japan). DNA was precipitated by adding 5 M NaCl and 100% ethanol and centrifuged at 21,900×g for 15 min. The DNA pellet was rinsed with 70% ethanol, dried, and dissolved in TE buffer. The purified DNA was quantified using a Biospec-nano (Shimadzu, Kyoto, Japan) and stored at − 80 °C until further analysis.

For DNA isolation, rumen fluid samples were thawed, and 250 μL aliquots were centrifuged at 9,700 × g for 30 min, after which the supernatant was discarded. For each sample, the pellet was re-suspended in 300 μL TE buffer, and total bacterial DNA was extracted as described previously (Morita et al., 2007 (link)) with minor modifications. The mixture was incubated with 750 μg/mL lysozyme (Sigma-Aldrich, St. Louis, MO, USA) at 37°C for 90 min. Then, 10 μL purified achromopeptidase (Wako Pure Chemical Industries, Ltd., Osaka, Japan) were added at a concentration of 10,000 U/mL and incubated at 37°C for 30 min. The suspension was treated with 60 μL 1% sodium dodecyl sulfate and 1 mg/mL proteinase K (Merck Japan, Tokyo, Japan), and incubated at 55°C for 5 min. The lysate was treated with phenol/chloroform/isoamyl alcohol (Wako Pure Chemical Industries, Ltd.) and chloroform (Life Technologies Japan, Ltd., Tokyo, Japan). DNA was precipitated by adding 5M NaCl and 100% ethanol and centrifuged at 21,900 × g for 15 min. The DNA pellet was rinsed with 70% ethanol, dried, and dissolved in TE buffer. The purified DNA was quantified using a Biospec-nano (Shimadzu, Kyoto, Japan) and stored at −80°C until further analysis.

Total bacterial DNA was extracted from rumen fluid samples as described by Kim et al. (21 (link)). Briefly, samples were incubated with 750 μg/mL lysozyme (Sigma-Aldrich Co., St. Louis, MO, USA) at 37°C for 90 min. This was followed by the addition of 10 μL of purified achromopeptidase (Wako Pure Chemical Industries Ltd., Osaka, Japan) at 10,000 U/mL, with the resulting mixture being incubated at 37°C for 30 min. This suspension was subsequently treated with 60 μL 1% sodium dodecyl sulfate and 1 mg/mL proteinase K (Merck Japan Ltd., Tokyo, Japan) with incubation at 55°C for 5 min, and the lysate thus obtained was extracted with three rounds of phenol/chloroform/isoamyl alcohol (Wako Pure Chemical Industries Ltd.) and chloroform (Life Technologies Japan Ltd., Tokyo, Japan). The resulting DNA was precipitated using 5 M sodium chloride and 100% ethanol, followed by centrifugation at 21,900 × g for 15 min at 4°C. The DNA pellet was rinsed with 70% ethanol, dried, and dissolved in Tris-hydrochloride buffer. The Purified DNA was quantified using a Biospec-nano spectrophotometer (Shimadzu Biotech, Kyoto, Japan) and stored at −80°C until used for further analyses.