title: GSM8876332: DOG 058; Canis lupus familiaris; OTHER
library_construction_protocol: Samples were transferred to a 2 ml tube containing 200 mg of 106/500μm glass beads (Sigma, St. Louis, MO) and 0.5 ml of Qiagen PM1 buffer (Valencia, CA). Mechanical lysis was performed for 20 minutes on a digital vortex mixer. After 5 min centrifugation, 0.45 ml of supernatants were aspirated and transferred to a new tube containing 0.15 ml of Qiagen IRS solution. The suspension was incubated at 4°C overnight. After 5 min centrifugation, the supernatant was aspirated and transferred to deep well plates containing 0.45 ml of Qiagen binding buffer supplemented with Qiagen ClearMag Beads. DNA was purified using the automated KingFisher™ Flex Purification System and eluted in DNase-free water. 12.5 ng of total DNA were amplified using universal primers targeting the V4 region of the bacterial 16S rRNA gene. Primer sequences contained overhang adapters appended to the 5' end of each primer for compatibility with the Illumina sequencing platform The primers used were F515/R806. Master mixes contained 12.5 ng of total DNA, 0.5 µM of each primer, and 2x KAPA HiFi HotStart ReadyMix (KAPA Biosystems, Wilmington, MA). The thermal profile for the amplification of each sample had an initial denaturing step at 95°C for 3 minutes, followed by cycling of denaturing at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and a 30-second extension at 72°C (25 cycles), a 5-minute extension at 72°C and a final hold at 4°C. Each 16S amplicon was purified using the AMPure XP reagent (Beckman Coulter, Indianapolis, IN). In the next step, each sample was amplified using a limited cycle PCR program, adding Illumina sequencing adapters and dual‐index barcodes (index 1(i7) and index 2(i5)) (Illumina, San Diego, CA) to the amplicon target. The thermal profile for the amplification of each sample had an initial denaturing step at 95°C for 3 minutes, followed by a denaturing cycle of 95°C for 30 seconds, annealing at 55°C for 30 seconds and a 30-second extension at 72°C (8 cycles), a 5-minute extension at 72°C and a final hold at 4°C. The final libraries were again purified using the AMPure XP reagent (Beckman Coulter), quantified, and normalized before pooling. The DNA library pool was then denatured with NaOH, diluted with hybridization buffer, and heat-denatured before loading on the MiSeq reagent cartridge (Illumina) and the MiSeq instrument (Illumina). Automated cluster generation and paired–end sequencing with dual reads were performed according to the manufacturer's instruction
identifiers.namespace: GEO
identifiers.id: GSM8876332_r1