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.