16S rRNA amplicon sequencing of gut microbiome

The gut digesta was immersed in liquid nitrogen for 10 min and then freeze-dried to remove the residual ethanol. DNA from each sample was extracted with a ZR Soil Microbe DNA MiniPrep kit, according to the manufacturer’s instructions. After quality and purity evaluation, the V3–V4 region of the 16S rRNA was amplified from the extracted DNA using the bacteria-specific primer pair 338F (forward: 5′-ACTCCTACGGGAGGCAGCAG-3′) (Mori et al., 2014 (link); Xu et al., 2016 (link)) and 806R (reverse: 5′-GGACTACHVGGGTWTCTAAT-3′) and the GeneAmp^®9700 thermal cycler (Applied Biosystems, Foster City, CA, United States) (Sun et al., 2014 (link)). The fragments (421–460 bp in length) were purified using the AxyPrep DNA gel extraction kit (Axygen Bio-sciences, Union City, CA, United States) and QuantiFluor^TM-ST fluorescence quantitative system (Promega, Madison, WI, United States). The purified amplicons were mixed at equimolar concentrations and then sequenced following a paired-end 250 bp × 2 strategy on an Illumina MiSeq platform operated by Majorbio Bio-Pharm Technology Co., Ltd., (Shanghai, China). The original reads were uploaded to the NCBI Sequence Read Archive database (Accession Number: PRJNA492364).