GSM9453351: WT mouse, normal diet, large intestine contents, sample 3; Mus musculus; OTHER
Approximately 100-200 mg of small intestine or large intestine contents from WD-fed or ND-fed MC4RKO and WT mice were freeze-dried using a VD-250R Freeze Dryer (TAITEC). The freeze-dried samples were homogenized at 1,500 rpm for 2 min using a multi-bead shocker (Yasui Kikai, Osaka, Japan). The DNA was extracted as follows: Lysis Solution F (Nippon Gene, Tokyo, Japan) was added to the crushed samples and incubated at 65 °C for 10 min. The samples were then centrifuged at 12,000 × g for 2 min, and the supernatant was collected. DNA was purified from the collected solution using the MPure-12 system and MPure Bacterial DNA Extraction Kit (MP Biomedicals, Tokyo, Japan). The concentration of the purified DNA was measured using Synergy LX (BioTek, Winooski, Vermont, USA) and QuantiFluor dsDNA System (Promega, Madison, WI, USA). The concentration of the purified DNA was measured using Synergy LX (BioTek, Winooski, Vermont, USA) and QuantiFluor dsDNA System (Promega, Madison, WI, USA). The sequencing library was prepared using a 2-step tailed PCR method. In the first PCR, 16S region-specific primers were used to amplify the V3-V4 region: 341f_MIX:5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNCCTACGGGNGGCWGCAG-3' 805r_MIX:5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNGACTACHVGGGTATCTAATCC-3' Each 10 μL reaction contained 0.05 U/μL of RxTaq HS (Takara Bio, Shiga, Japan), 0.5 μM of each primer, and 1 ng of template DNA. The PCR conditions were as follows: initial denaturation at 94°C for 2 min; 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s; followed by a final extension at 72°C for 5 min. PCR products were cleaned using AMPure XP beads (Beckman Coulter, CA, USA) following the manufacturer's protocol. A second PCR was performed using the following indexed primers: F:5'-AATGATACGGCGACCACCGAGATCTACAC-Index2 ACACTCTTTCCCTACACGACGC-3' R:5'-CAAGCAGAAGACGGCATACGAGAT-Index1 GTGACTGGAGTTCAGACGTGTG-3' In this step, cleaned amplicons from the first PCR served as templates in 10 μL reactions with 0.05 U/μL of RxTaq HS (Takara Bio) and 0.5 μM of each indexed primer. Amplification was conducted for 10 cycles under the same cycling conditions as the first PCR. The concentration of the library was measured using Synergy H1 (BioTek) and QuantiFluor dsDNA System. Library quality was assessed using a Fragment Analyzer and a dsDNA 915 Reagent Kit (Advanced Analytical Technologies, Orangeburg, NY, USA).