title: 16S high throughput for different MPs treatments
design: The hypervariable region V3-V4 of the bacterial 16S rRNA gene were amplified using the forward primer 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and the reverse primer 806R (5'-GGACTACHVGGGTWTCTAAT-3') by an ABI GeneAmp 9700 PCR thermocycler (ABI, CA, USA). The PCR reaction mixture included 4 L of 5  Fast Pfu buffer, 2 L of 2.5 mM dNTPs, 0.8 L of each primer (5 M), 0.4 L of Fast Pfu polymerase, 10 ng of template DNA, and ddH2O to reach a final volume of 20 L. The PCR amplification cycling conditions were as follows: an initial denaturation at 95 for 3 min, followed by 27 cycles of denaturing at 95 for 30 s, annealing at 55 for 30 s, and extension at 72 for 45 s, with a final extension at 72  for 10 min, followed by cooling to 10. All samples were amplified in triplicate. The PCR product was extracted from a 2% agarose gel, purified, and then quantified using Quantus Fluorometer (Promega, USA). Purified amplicons were pooled in equimolar amounts and subjected to 300-bp paired-end sequencing according to the standard protocols on an Illumina MiSeq platform (Illumina, San  Diego, USA) in Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China).