2.7 Gut microbiota analysis
Gut microbiota was examined as before by 16S ribosomal RNA analysis on the Illumina MiSeq platform (Illumina, San Diego, USA) according to standard protocols (Shao et al., 2019). DNA was extracted from colon contents using an Omega Mag-Bind Soil DNA Kit (200) (Omega Bio-Tek, USA). Purified PCR products were prepared using Q5® High-Fidelity DNA Polymerase (NEB, USA) and products were quantified, then each PCR sample was diluted 5 times to 20 ng/μL. PCR Amplification System: PCR mixed product sample (2 µL), 5× reaction buffer (5 μL), 5× GC buffer (5 μL), dNTP (2.5 mM 2 μL), Forward primer (10 µM, 1 μL), Reverse primer (10 µM, 1 μL), Q5 DNA Polymerase (0.25 μL), DNA template (2 μL), ddH2O 8.75 μL. PCR amplification of the 16S rRNA genes V3–V4 region was performed using the forward primer 338F 5’-ACTCCTACGGGAGGCAGCA-3’ and reverse primer 806R 5’-GGACTACHVGGGTWTCTAAT-3’. Sample-specific 7-bp barcodes were incorporated into the primers for multiplex sequencing. The PCR components contained 5 μl of Q5 reaction buffer (5×), 5 μl of Q5.High-Fidelity GC buffer (5×), 0.25 μl of Q5 High-Fidelity DNA Polymerase (5U/μl), 2 μl (2.5 mM) of dNTPs, 1 μl (10 uM) of each Forward and Reverse primer, 2 μl of DNA Template, and 8.75 μl of ddH2O. Thermal cycling consisted of initial denaturation at 98 °C for 2 min, followed by 25 cycles consisting of denaturation at 98 °C for 15 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s, with a final extension of 5 min at 72 °C. The amplicon library was then used for paired-end sequenced (2 × 250bp) on an Illumina MiSeq platform (Illumina, San Diego,USA) according to standard protocols.