Disruptor genie instrument

Fecal samples were collected from the mice who were fed the AIN-76A-based modified diet for two weeks. DNA was extracted from ≤100 mg of feces using the ZymoBIOMICS DNA Miniprep kit (Zymo Research, Irvine, CA, USA), according to the manufacturer’s protocol. A Disruptor Genie instrument (Scientific Industries, Bohemia, NY, USA) was used for bead beating, for 20 min, at maximum speed.

Feces were collected from mice administered PEG for two or four weeks. Feces were immediately frozen in liquid nitrogen and stored in a deep freezer (−80 °C), as storage at room temperature for more than 15 min or in a domestic freezer for more than three days can impact the composition of bacterial taxa in meta 16S analysis [19 (link)]. DNA was extracted from a pellet of feces (≤50 mg) using the ZymoBIOMICS DNA Miniprep kit (Zymo Research, Irvine, CA, USA) following the manufacturer’s protocol. Bead beating was performed for 20 min using a Disruptor Genie instrument (Scientific Industries, Bohemia, NY, USA) at maximum speed.

Three representative chicken cecum samples were selected at each time point, to exclude the samples with maximum and minimum bacterial counts, and subjected to 16S rRNA sequencing analysis. Aliquots of the BPW suspensions (1 ml) were centrifuged at 21,500 × g for 10 min at 4°C. The pellets were then resuspended in 400 μl of homogenization solution containing 2 μl of proteinase K (Promega, Madison, WI, USA). After incubation at 37°C for 10 min, the samples were vortexed for 5 min with Zirconia beads (ZircoPrep Mini; Nippon Genetics, Tokyo, Japan) on a Disruptor Genie instrument (Scientific Industries, Bohemia, NY, USA). After centrifugation at 11,000 × g for 5 min, 100 μl of each supernatant were transferred into 300 μl of lysis buffer (Promega). DNA extraction was then carried out using a Maxwell Blood DNA kit in a Maxwell RSC instrument (Promega). The concentration and quality of the extracted DNA were measured on a Tape Station 4150 system (Agilent Technologies, Santa Clara, CA, USA), and the samples were stored at −80°C until use.