title: GSM9086293: Trmt6 KO rep1; Mus musculus; OTHER
library_construction_protocol: Microbial DNA was extracted from fecal samples using an E.Z.N.A.® soil DNA Kit (Omega Bio-tek, Norcross, GA), according to the manufacturer 's protocols. The V3-V4 hypervariable regions of the bacterial 16S rRNA gene were amplified with the primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') by a thermocycler PCR system (GeneAmp 9700, ABI, Foster, CA, United States). The PCR reactions were as follows: denaturation at 95°C for 3 min, 27 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, elongation at 72°C for 45 s, extension at 72°C for 10 min, and ending at 4°C. Each 20 μL reaction mixture contained 4 μL of 5× TransStart FastPfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL TransStart FastPfu DNA Polymerase, and 10 ng template DNA. PCR was performed in triplicate. The PCR product was extracted from 2% agarose gel and purified using an AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA), according to the manufacturer's instructions, and quantified using Qubit 4 (Thermo Fisher, United States).