Quantifluortm st fluorometer

The V1–V3 region of the 16S rDNA gene was amplified with barcoded primers (8F/533R) (Wu et al., 2012 (link)). Replicated PCR products of each sample were pooled and separated by 2% agarose gel electrophoresis, then purified with a DNA gel extraction kit (Axygen, China). Prior to sequencing, the concentrations of purified amplicon DNA were determined using a QuantiFluorTM-ST Fluorometer (Promega, Madison, WI, United States). Equal amounts of the PCR products were mixed together and sequenced on a 454 GS FLX + platform (Roche Applied Science, Indianapolis, IN, United States) at Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China.

According to the manufacturer’s instructions, we used the FastDNA™ SPIN Kit for Soil (MP 112 Biomedicals, Solon, OH, USA) to extract DNA from 0.5 g fresh soil samples. The quality of the DNA extracted was examined by 1% (w/v) agarose gel electrophoresis. Additionally, the concentrations were measured by using a UV–vis spectrophotometer (NanoDrop 2000, Thermoscientific, Waltham, MA, USA). The V3–V4 regions of 16S rRNA genes of the bacteria were amplified using the primer set 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). Amplification conditions are as follows: 2 min at 95 °C, 30 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s, and a final elongation for 5 min in a GeneAmp^® 9700 Thermo Cycler (ABI, Foster City, CA, USA). The triplicate amplicons were pooled together, electrophoresed on a 2% (w/v) agarose gel, and recovered through an AxyPrep DNA Gel Extraction Kit (AXYGEN, Hangzhou, China). This purified amplicon was quantified using a QuantiFluor^TM-ST Fluorometer (Promega, Madison, WI, USA). Then a composite sequencing library was constructed by combining equimolar ratios of amplicons from all the 17 samples. Finally, the sample libraries were analyzed by using the Illumina Miseq platform (Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China) [35 (link)].

Purified PCR products from all tests were quantified using Quant-it PicoGreen dsDNA Assay Kit (Invitrogen) in a Light Cycler 480 II real-time PCR machine (Roche). Then they were adjusted to equimolar concentration (2x105 (link) molecules/μl in TE buffer) and all amplification products were pooled together. The pool was then quantified using the Quant-it PicoGreen dsDNA Assay Kit (Invitrogen) on a QuantiFluor^TM-ST fluorometer (Promega, US).
Emulsion PCR was performed according to the manufacturer's instructions with GS Junior Titanium emPCR Kit Lib-A (Roche) and sequenced in a single 454 Roche Junior run.

Total genomic DNA of cecal contents was extracted by a Stool DNA Kit (Omega Bio-TEK, Norcross, GA, USA) according to the manufacturer’s procedure. The quantity of DNA was analyzed by a NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and the integrity of DNA was checked by 1% agarose gel electrophoresis. The V3-V4 hypervariable region of the 16S rRNA gene was amplified via the universal primers 338F (5′-barcode-ACTCCTRCGGGAGGCAGCAG-3′) and 806R (5′-GGACTACCVGGGTATCTAAT-3′). Amplicons were extracted from 2% agarose gels, purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and quantified with a QuantiFluor TM-ST fluorometer (Promega, Madison, WI, USA). Purified amplicons were pooled and then paired-end sequenced using the Illumina MiSeq platform (Illumina, San Diego, CA, USA). Demultiplexing and quality filtering of raw sequences were performed by QIIME (version 1.17). Operational taxonomic units (OTU) were clustered using UPARSE software according to a similarity of more than 97%. The taxonomy of 16S rRNA gene sequences was determined by the RDP Classifier (http://rdp.cme.msu.edu/ accessed on 10 November 2021) with a confidence threshold of 70%. Data analysis and processing were conducted based on the Majorbio cloud platform (Majorbio Bio-pharm Technology Co., Ltd., Shanghai, China).