Purified achromopeptidase

Stool samples collected from mice were immediately frozen using liquid nitrogen and stored at −80 °C until use. Bacterial genomic DNA was isolated as described previously with modifications51 (link)52 (link). The bacterial pellet was suspended and incubated with lysozyme (15 mg/ml, Wako Pure Chemical Industries, Ltd) at 37 °C for 1 h in 100 mM Tris-HCl/10 mM EDTA (10 × TE). Purified achromopeptidase (Wako Pure Chemical Industries, Ltd) was added at a final concentration of 2000 U/ml and then incubated at 37 °C for 30 min. The suspension was treated with 1% (wt/vol) sodium dodecyl sulfate (Wako Pure Chemical Industries, Ltd) and proteinase K (1 mg/ml, Merck Japan, Tokyo, Japan) and incubated at 55 °C for 1 h. The lysate was treated with phenol/chloroform/isoamyl alcohol, and DNA in the aqueous phase was precipitated by the addition of ethanol and pelleted via centrifugation at 5,000 × g at 4 °C for 15 min. The DNA pellet was rinsed with 75% ethanol, dried, and dissolved in 1 × TE. DNA samples were purified by treatment with RNase A (1 mg/ml, Wako Pure Chemical Industries, Ltd) at 37 °C for 30 min and precipitated by the addition of equal volumes of a 10% polyethylene glycol solution (PEG6000-2.5 M NaCl). DNA was pelleted via centrifugation at 18,000 × g at 4 °C for 10 min, rinsed with 75% ethanol, and dissolved in 1 × TE.

Bacterial DNA was extracted as described previously (21 (link)). After thawing, 2 ml of uterine and vaginal samples were mixed with 20 ml of sterile saline solution and centrifuged at 8,000 g for 15 min. Pellets were then suspended in 800 μl 10 mM Tris-HCl and 10 mM EDTA buffer containing lysozyme (Sigma-Aldrich Co., LCC, Missouri, USA) (Final concentration: 15 mg/ml). Mixtures were incubated at 37°C for 1 h. Purified achromopeptidase (Wako Pure Chemical Inc., Osaka, Japan) (Final concentration: 2,000 U/ml) was added and incubated 37°C for 30 min. Finally, proteinase K (Takara-Bio Inc., Shiga, Japan) (Final concentration: 1 mg/ml) with 20% sodium dodecyl sulfate (Sigma-Aldrich Co., LCC) was added and incubated at 55°C for 1 h. The DNA was isolated with phenol:chloroform:isoamyl alcohol (25:24:1, v/v), washed twice with 75% ethanol, and dissolved in 100 μl TE buffer. RNase A (Nippon Gene Co., Ltd., Tokyo, Japan) (Final concentration: 0.1 mg/ml) was added to the mixture, and incubated at 37°C for 1 h. Subsequently, the DNA was purified using a High Pure PCR Template Kit (Roche Inc., Basel, Switzerland) according to the manufacturer's instructions. Elution was performed in 50 μl of TE buffer and then samples were stored at −20°C until further analysis.

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.