Phenol chloroform isoamyl alcohol

Tris-HCl at 1 M (pH 9.0), ethylenediaminetetraacetic acid (EDTA) at 0.5 M (pH 8.0), Tris-EDTA buffer, Tris-EDTA-saturated phenol, phenol/chloroform/isoamyl alcohol (25:24:1), sodium acetate at 3 M (pH 5.2), and 10% sodium dodecyl sulfate were obtained from Nippon Gene Co., Ltd. (Tokyo, Japan). Isopropanol, ethanol, sucrose, glycerol, Tris, EDTA-2K, potassium dihydrogen phosphate, and potassium hydrogen phosphate were obtained from Fujifilm Wako Pure Chemical Co. (Osaka, Japan).

Approximately 30 mg of stool samples were homogenized in 1 mL extraction buffer (10% 400 µL sodium dodecyl sulfate (Sigma-Aldrich Japan Inc., Tokyo, Japan) in Tris-EDTA (TE) buffer (pH 7.4 10 mmol / L Tris ; Fujifilm Waco Pure Chemical Corporation, Osaka, Japan and pH 8.0 1 mmol / L EDTA ; Fujifilm Waco Pure Chemical Corporation), 200 µL 3 M sodium acetate (Fujifilm Waco Pure Chemical Corporation), and 400 μL phenol : chloroform : isoamyl alcohol (25 : 24 : 1 v / v) (Nippon Gene, Tokyo, Japan)), added to a Lysing Matrix E Tube (MP Biomedicals, Solon, OH, USA), and homogenized using a FastPrep-24 automated cell disruptor (MP Biomedicals) for 40 sec at 6 m / sec. This procedure was performed twice. The homogenate was centrifuged for 30 min at 10,000 × g, and an extract of DNA was collected as the aqueous phase and purified by adding a matched volume of phenol : chloroform : isoamyl alcohol (25 : 24 : 1, v / v) twice. Next, an equal volume of isopropyl alcohol (Fujifilm Waco Pure Chemical Corporation) was added, and DNA was obtained as a pellet by centrifugation (5 min, 10,000 × g). After drying, the DNA was dissolved in TE. The preparation of the 16S rRNA gene metagenome library for MiSeq (Illumina, Inc., San Diego, CA, USA) was conducted in accordance with the manufacturer's instructions. Sequence data were processed using the 16S rRNA sequence analysis pipeline QIIME 1.8.0.