16S rRNA Amplification and Sequencing of Gut Microbiome

Metagenomic DNA was extracted from each of the 36 samples from ileum, colon and rectum using DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The V3-V4 region of the bacterial 16S rRNA genes was amplified by PCR, using specific barcode primers 338 F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806 R (5′-GGACTACHVGGGTWTCTAAT-3′) as described by Bi et al., [25 (link)]. The PCR was carried out by the following protocol: an initial denaturation step at 95 °C for 2 min, followed by 30 cycles at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s and a final extension at 72 °C for 5 min. The PCR products were visualized on 2% agarose gels and were quantitatively determined using QuantiFluor-ST Fluoremeter (Promega, Wisconsin, USA) and PicoGreen dsDNA Quantitation Reagent (Invitrogen, Carlsbad, CA, USA). The PCR amplicons were purified with an AxyPrep DNA Purification kit (Axygen Biosciences, Union City, CA, USA).