Axyprep dna purification kit

Amplicons were detected with 2% agarose gel electrophoresis and purified with the AxyPrep DNA Purification Kit (Axygen Biosciences, Union City, CA, USA). PCR products were then visualized on agarose gels and were determined quantitatively with PicoGreen dsDNA Quantitation Reagent (Invitrogen, Carlsbad, USA) and QuantiFluor-ST Fluoremeter (Promega, USA). Purified amplicons were pooled with an Illumina MiSeq platform (Majorbio, Shanghai, China) following the standard protocols in equimolar and paired-end sequenced (2 × 300). The raw data were uploaded to NCBI SRA Database, with the SRA accession: SRP111417.

Soil total DNA was extracted using a MOBIO-12888 soil DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA, United States). DNA quantity and quality were first detected by a garose gel electrophoresis (1%) and then detected by using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific Waltham, MA, United States). The special primers ACIDO (5′GCTCAGAATSAACGCTGG3′)/342r(5′CTGCTGCSYCCCGTAG3′) (~336 bp) were selected to amplify the Acidobacterial region (Lee and Cho, 2011 (link)). The PCR amplification system was conducted in a 25 μl reaction systems, consisted of 12.5 μl of PCR mix (Invitrogen, Shanghai, China), 1.5 μl of forward and reverse primers (10 μmol·L⁻¹), 1 μl of DNA template (100 ng·L⁻¹), and enough ultrapure water (ddH₂O) to reach a 25 μl reaction volume. The amplification program were the following: pre-denaturation at 95°C for 8 min, 30 cycles of denaturation at 95°C for 60 s, annealing at 55°C for 60 s, and extension at 72°C for 60 s, and finally an extension step at 72°C for 20 min. The PCR products were inspected by 2% agarose electrophoresis, and the PCR products were purified with the AxyPrep DNA purification kit (Axygen Biosciences, Union City, CA, United States). Three independent PCR replicates per sample and then three PCR samples were pooled at equal amounts and paired-end sequenced on the Illumina MiSeq v3 platform (2 × 300 bp).

According to the procedure suggested by manufacturer, the cecal microbial community DNA was extracted using QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany). Bacterial universal primers, 338F (50-ACTCCTACGGGAGGCAGCAG-30) and 806R (50-GGACTACHVGGGTWTCTAAT-30) were used to amplify along with V3/V4 hypervariable regions of 16s rDNA by Polymerase Chain Reaction (PCR) [32 (link)]. The annealing temperature was 55 °C during the 30 PCR cycles. The fungal ITS gene amplification was conducted using primers ITS5F (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and ITS2R (5′-GCTGCGTTCTTCATCGATGC-3′). Visualized the PCR products by 1.5% agarose gel electrophoresis and then quantified it by PicoGreen dsDNA Quantitation Reagent (Invitrogen, USA). To obtain purified PCR products, the AxyPrep DNA Purification kit was involved in this part (Axygen Biosciences, USA). The library generated by the purified PCR products were Paired end sequenced (Paired_End) based on the Illumina NovaSeq sequencing platform (Illumina NovaSeq PE250, United States). We spliced and filtered the original data to filter out contaminated data, such as chimera sequences, nucleotide mismatch, and ambiguous character reads, to obtain accurate and reliable adequate data.

DNA extraction was performed as previously described (Hu et al., 2012a (link)). The extracted genomic DNA was examined in 1.0% agarose gels by electrophoresis and quantified using a NanoDrop ND-1000 spectrophotometer as previously introduced (Hu et al., 2014a (link)). The DNA was stored at −20°C for further PCR amplification, and the primer pairs 357F and 926R were used to amplify the V3-V5 hypervariable regions of bacterial 16S rRNAs (Escherichia coli positions 357–926) (Liu et al., 2013a (link),b (link)). A barcode was permuted for each sample to allow for the identification of individual samples in a mixture within a single pyrosequencing run (Hu et al., 2014b (link)). Each sample was amplified in triplicate with a 20 μ L reaction system using the following protocol: 95°C for 2 min, 25 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, and a final extension at 72°C for 10 min. The three replicate PCR products of each sample were mixed together and purified with an AxyPrep DNA purification kit (AXYGEN). All of the samples were quantified by TBS-380 and mixed at an equimolar ratio in a single tube to be run on a Roche FLX 454 pyrosequencing machine (Roche Diagnostics Corporation, Branford, CT, USA), which produces reads from the forward direction primer 357F.

PCR products were detected using 2% agarose gels electrophoresis, purified with AxyPrep DNA Purification kit (Axygen Biosciences, Union City, USA). The PCR products were visualized on agarose gels and were quantitatively determined using QuantiFluor-ST Fluoremeter (Promega, Wisconsin, USA) and PicoGreen dsDNA Quantitation Reagent (Invitrogen, Carlsbad, USA). Purified amplicons were pooled in equimolar and paired-end sequenced (2 × 300) on an Illumina MiSeq platform (Allwegene, Beijing, China) according to the standard protocols. And the raw data were uploaded to NCBI SRA Database, with the SRA accession: SRP090426.

Soil total DNA was extracted using the Power Soil DNA isolation kit 12888 according to the manufacturer's instructions. Using 50 ng DNA as a template, the 338F (5′‐ACT CCT ACG GGA GGC AGC A‐3′) and 806R (5′‐GGA CTA CHV GGG TWT CTA AT‐3′) universal primers were used to amplify the V3–V4 regions (Liu et al., 2016 (link)). The PCR amplification system consisted of 12.5 μl PCR mix, 1.2 μl forward and reverse primers (5 μmol·L⁻¹), 1 μl DNA template (50 ng·L⁻¹), and enough ultrapure water (ddH₂O) to reach a 25 μl reaction volume. The amplification conditions as follows: pre‐denaturation at 95°C for 5 min, 35 cycles of denaturation at 95°C for 30 s, annealing at 52°C for 45 s, extension at 60°C for 60 s, and finally an extension step at 72°C for 10 min. The PCR products were inspected using 2% agarose electrophoresis, and the PCR amplicons were purified with the AxyPrep DNA purification kit (MAG‐PCR‐CL‐5, Axygen). Three independent PCR replicates were produced per sample then pooled in equal amounts; the pooled samples were then paired‐end sequenced on the Illumina MiSeq v3 platform (2 × 300 bp). The raw sequences were uploaded to the NCBI Sequence Read Archive database under accession number PRJNA691134.

The ABI GeneAmp 9700 PCR Thermocycler (ABI, CA, USA) was utilized to amplify the V3-V4 hypervariable region of the bacterial 16S rRNA gene. PCR began with initial denaturation for 3 min at 95 °C, followed by 27 cycles of 30 s at 95 °C, an annealing for 30 s at 55 °C, extension for 45 s at 72 °C, and a single extension for 10 min at 72 °C. The PCR products were purified using the AxyPrep DNA Purification Kit (Axygen Biosciences, CA, USA), which were measured using 2% agarose gel electrophoresis. Subsequently, the PCR products were quantified by QuantiFluor-ST Fluorometer (Promega, Madison, WI, USA), and paired-end sequencing (2 × 300) of purified and pooled amplicon libraries was performed on the Illumina MiSeq platform (Illumina, San Diego, USA) based on the standard protocol of Majorbio Bio-Pharm Technology Co., Ltd., (Shanghai, China).