Quantifluor st blue fluorescence quantitative system

The DNA extraction was performed according to the kit instructions (D4015, Omega Inc., Norcross, GA, United States), and the quality of the extracted DNA was detected by 1% agarose gel electrophoresis. The 16S rRNA gene (V3-V4 region) was amplified using the specific primers 799F (5′-AACMGGATTAGATACCCKG-3′) and 1193R (5′- ACGTCATCCCCACCTTCC-3′) with sample-specific barcodes. The bacterial 16S rDNA gene fragment was amplified by PCR (GeneAmp®9700; ABI, America). Each sample was replicated 3 times. The PCR products of the same sample were mixed and detected by 2% agarose gel electrophoresis. The AxyPrep DNA gel recovery kit (AXYGEN Company) was used to cut the gel to recover the PCR products, and Tris HCl was used for elution. Next, the PCR products were detected and quantified by QuantiFluor™-ST blue fluorescence quantitative system (Promega Company) and then mixed in the corresponding proportion according to the requirement of sequencing quantity of each sample. Finally, the PCR products were denatured with sodium hydroxide to generate single-stranded DNA fragments sequenced on the Illumina platform 250PE.

The V5–V7 region of bacterial 16S ribosomal RNA (rRNA) gene was amplified through polymerase chain reaction (PCR) using universal primers: 799F (5′-AACMGGATTAGATACCCKG-3′) and 1193R (5′-ACGTCATCCCCACCTTCC-3′), and the barcode sequence, provided by Majorbio Biotechnology Co. Ltd. (Shanghai, China) was added to the 5′ end of the PCR product. The PCR reaction system was 50 μL, with 50 ng of total DNA from tomato root template, 1.5 μL of 10 μmol/L of 5′ and 3′ end primers respectively, 5 μL of 2 mmol/L dNTPs, 2 μL of MgSO₄, 5 μL of 10× KOD buffer, 1 μL of KOD Plus, and added ddH₂O to 20 μL. PCR reaction conditions were as follows: 95°C for 3 min; 30 cycles at 95°C for 30 s, 63°C for 30 s, 68°C for 30 s; and finally 68°C for 5 min. Each sample was repeatedly amplified by PCR thrice, and the three PCR products were mixed. The PCR products were cut and purified using an AxyPrepDNA gel recovery kit (Axygen Biosciences, Union City, CA, USA), and the DNA concentration was detected using 2% agarose electrophoresis. PCR products were detected and quantified using the QuantiFluor™-ST blue fluorescence quantitative system (Promega, Madison, WI, USA) and mixed according to the sequencing quantity of each sample. Majorbio Biotechnology Co. Ltd. (Shanghai, China) generated libraries for sequencing on Miseq PE300 (Illumina, San Diego, CA, United States).

The genomic DNA of the enrichments was extracted using the FastDNA SPIN Kit (MP Biomedicals, Santa Ana, United States) and determined with a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States). The bacterial or archaeal 16S rRNA fragment covering the V3 and V4 variable regions was amplified with the primer pair 338F (5′-actcctacgggaggcagca-3′)/806R (5′-ggactachvgggtwtctaat-3′) (Zakrzewski et al., 2012 (link); Klindworth et al., 2013 (link)). Unique 5–8 bp barcodes (Supplementary Table S1) for different samples were incorporated into the primers for multiplex sequencing (Parameswaran et al., 2007 (link)). The PCR system and amplification conditions were similar to those described by Mao et al. (2015) (link). The PCR products were quantified with a QuantiFluor-ST blue fluorescence quantitative system (Promega, United States). The amplicon library was paired-end sequenced (2 × 300) on an Illumina MiSeq platform according to the standard protocols (Shanghai BIOZERON Co., Ltd., China). The raw sequencing data were deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under the accession number PRJNA498798.

The faeces of the rats in the blank, model, and SMYH groups were collected, with 10 rats in each group, which were collected from the last 3 days of the experiment and stored at −80°C. The CTAB or SDS method was used to extract genomic DNA from rat intestinal faeces. According to the sequencing region selected, specific primers with barcodes and high-fidelity DNA polymerase were used for PCR amplification of the selected V3-V4 variable regions. A QuantiFluor™-ST blue fluorescence quantitative system (Promega) was used to detect and quantify the PCR-amplified products. According to the sequencing volume requirements of each sample, the corresponding mixing ratio was determined. An NEB Next® Ultra™ DNA Library Prep Kit was used for library construction. The constructed library was subjected to quality inspection using an Agilent Bioanalyzer 2100 and Qubit. After the library quality inspection was performed, the library was analysed on a computer.

The total DNA of each sample was isolated from 0.25 g of soils using the PowerSoil DNA Isolation Kit (MoBio Laboratories, Inc., Carlsbad, CA, United States). Prior to amplification, the DNA concentration was standardized to be the same. The 16S rRNA gene’s V3-V4 region was chosen to create the community library using the forward primers 338\u00B0F (Huse et al., 2008 (link)) and reverse primer 806 R (Li et al., 2019 (link)), using a six-base barcode that is particular to each sample. Supplementary Table S2 offers more information on the PCR conditions. PCR products from all samples were combined and identified by 2% agarose gel electrophoresis, and the PCR products were recovered by gel cutting using the AxyPrep DNA Gel Recovery Kit (AXYGEN). PCR products were quantitatively detected by QuantiFluor™-ST Blue fluorescence quantitative system (Promega), and the MiSeq sequencing library was constructed using TruSeqTM DNA Sample Prep Kit, which was sequenced based on PE300 strategy. The PCR products were purified, pooled in equimolar amounts, and paired-end sequenced (2 × 300) using an Illumina MiSeq platform at Shanghai Meiji Biological Medicine Technology Co Ltd., Shanghai, China.